Chimeric polypeptides and methods of altering the membrane localization of the same

ABSTRACT

Methods and compositions are provided for reversibly localizing proteins to the exterior part of the cell surface. Compositions provided herein can include nucleic acids that encode polypeptides of interest and the ligand binding domain (LBD) of a nuclear hormone receptor. Medical applications are provided, including controlling the toxicity of CAR T cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 62/477,733, filed Mar. 28, 2017, and U.S. Provisional PatentApplication Ser. No. 62/587,262, filed Nov. 16, 2017; the entirecontents of which are herein incorporated by reference.

TECHNICAL FIELD

The present disclosure relates to the field of biotechnology, and morespecifically, to the regulation of the cellular localization of achimeric polypeptide.

BACKGROUND

Chimeric antigen receptors (CARs) are a subclass of single-chain andmulti-chain polypeptides. Chimeric antigen receptors typically include,e.g., an antigen-binding domain, a transmembrane domain, aco-stimulatory domain, and a CD3ζ or other signaling domain. CARs havebeen investigated for use in the clinical treatment of differentcancers, e.g., hematological cancers, in mammals with considerablesuccess. A limiting factor, however, has been uncontrollable toxicityassociated with CAR anticancer activity.

Nuclear hormone receptors are class of polypeptides that include severaldomains, including a DNA-binding motif and a ligand-binding domain. Inthe absence of ligand, naturally-occurring nuclear hormone receptorsassociate with various chaperones and other interacting proteins whichsequester the nuclear hormone receptor outside of the nucleus. Ligandbinding induces changes in nuclear hormone receptor conformation andinteraction partners and allows the nuclear translocation andtranscriptional activation or repression of nuclear hormone targetgenes. While the classical view of steroid signaling holds that thesehormones bind intracellular receptors that act as ligand-activatedtranscription factors, it has been reported that steroids may also actby a non-genomic mechanism at the cell surface.

SUMMARY

The present disclosure is based, at least in part, on the discovery thatthe reversible extracellular membrane localization of chimericpolypeptides that include a transmembrane domain can be utilized formedical applications where it is desired to have reversibly expressed onthe extracellular membrane a protein or peptide of interest. Anexemplary feature of the present disclosure is that the toxicityassociated with CAR T cells can be regulated by reversibly expressing ontheir surface an antigen binding domain of a CAR.

The present disclosure is further based, at least in part, on thediscovery that the plasma membrane localization or extracellularmembrane localization of chimeric polypeptides that include atransmembrane domain and a hormone receptor ligand binding domain, wherethe transmembrane domain and the hormone receptor ligand binding domaindirectly abut each other or are separated by 1 to about 700 amino acids(e.g., 1 to about 500 amino acids), and the transmembrane domain and thehormone receptor ligand binding domain are not both present in the sameendogenous single-chain polypeptide in a mammal (e.g., a single-chainchimeric antigen receptor), in a mammalian cell can be regulated bycontacting the mammalian cell with a hormone or a hormone analogue. Inview of this discovery, provided herein are: single-chain chimericpolypeptides, single-chain chimeric antigen receptors, and multi-chainchimeric antigen receptors; nucleic acids encoding the same; vectorsincluding any of these nucleic acids; mammalian cells including any ofthese vectors; and methods of reversibly inducing the extracellularmembrane localization and methods of altering the membrane localizationof these single-chain chimeric polypeptides, single-chain chimericantigen receptors, and multi-chain chimeric polypeptides.

In some embodiments, reversibly inducing the plasma membranelocalization or extracellular membrane localization of polypeptides(e.g., single-chain chimeric polypeptides, single-chain chimeric antigenreceptors, and/or multi-chain chimeric antigen receptors) using one ormore of the compositions or methods disclosed herein is useful in thearea of cell-based adoptive immunotherapy, in which immune cellsisolated from a subject can be modified to express synthetic proteinsthat enable the cells to perform new therapeutic functions after theyare subsequently transferred back into the subject. Non-limitingexamples of such synthetic proteins are chimeric antigen receptors(CARs) and engineered T cell Receptors (TCRs).

In some embodiments, reversibly inducing the plasma membranelocalization or extracellular membrane localization of polypeptides(e.g., single-chain chimeric polypeptides, single-chain chimeric antigenreceptors, and/or multi-chain chimeric antigen receptors) using one ormore of the compositions or methods disclosed herein is useful incontrolling the toxic effects of chimeric antigen T cells (CAR-T cells)involved in eliminating cancer cells by regulating the presence orabsence of an antigen binding moiety on the plasma membrane orextracellular membrane of the cell.

Provided herein are single-chain chimeric polypeptides that include: atransmembrane domain; and a hormone receptor ligand binding domain,where: the transmembrane domain and the hormone receptor ligand bindingdomain directly abut each other or are separated by 1 to 500 aminoacids; and the transmembrane domain and the hormone receptor ligandbinding domain are not both present in the same endogenous single-chainpolypeptide in a mammal. In some embodiments of any of the single-chainchimeric polypeptides described herein, the transmembrane domain is atransmembrane domain from: α chain of a T cell receptor, β chain of theT cell receptor, ζ chain of the T cell receptor, CD28, CD3ε, CD3δ, CD3γ,CD33, CD37, CD64, CD80, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD86,CD134, CD137, or CD154. In some embodiments of any of the single-chainchimeric polypeptides described herein, the hormone receptor ligandbinding domain is a ligand binding domain from an estrogen receptor, aprogesterone receptor, or an androgen receptor. In some embodiments ofany of the single-chain chimeric polypeptides described herein, thehormone receptor ligand binding domain is a ligand binding domain froman estrogen receptor.

In some embodiments of any of the single-chain chimeric polypeptidesdescribed herein, the transmembrane domain and the hormone receptorligand binding domain directly abut each other. In some embodiments ofany of the single-chain chimeric polypeptides described herein, thetransmembrane domain and the hormone receptor ligand binding domain areseparated by 1 to 500 amino acids (e.g., 1 to 250 amino acids, or about1 to 50 amino acids).

Also provided herein are nucleic acids that include a nucleotidesequence encoding any of the single-chain chimeric polypeptidesdescribed herein. Also provided herein are vectors that include any ofthe nucleic acids that include a nucleotide sequence encoding any of thesingle-chain chimeric polypeptides described herein. Also providedherein are mammalian cells that include any of the vectors describedherein.

Also provided herein are methods of inducing the membrane localizationof a single-chain chimeric polypeptide in a mammalian cell that include:contacting a mammalian cell expressing any of the single-chain chimericpolypeptides described herein with an amount of a hormone or a hormoneanalogue sufficient to induce localization of the single-chain chimericpolypeptide to the extracellular side of the plasma membrane of thecell.

Also provided are methods of reversibly altering the membranelocalization of a single-chain chimeric polypeptide in a mammalian cellthat include: (a) contacting a mammalian cell expressing any of thesingle-chain chimeric polypeptides described herein with an amount of ahormone or a hormone analogue sufficient to induce localization of thesingle-chain chimeric polypeptide to the extracellular side of theplasma membrane of the cell; and (b) contacting the mammalian cell witha reduced amount of the hormone or the hormone analogue that results ina decreased level of the single-chain chimeric polypeptide on theextracellular side of the plasma membrane of the mammalian cell. Someembodiments of any of the methods described herein further include: (c)contacting the mammalian cell with an increased amount of the hormone orthe hormone analogue that results in an increased level of thesingle-chain chimeric polypeptide on the extracellular side of theplasma membrane of the cell as compared to the level of in step (b).

Some embodiments of any of the methods described herein further include:introducing a nucleic acid encoding the single-chain chimericpolypeptide into a mammalian cell to generate the mammalian cellexpressing the single-chain chimeric polypeptide. In some embodiments ofany of the methods described herein, the mammalian cell is a T cell(e.g., a T-cell selected from the group of a CD8⁺ T cell, a CD4⁺ T cell,a memory T cell, a Treg cell, and natural killer T cell).

In some embodiments of any of the methods described herein, themammalian cell is a mammalian cell previously obtained from a subject.In some embodiments of any of the methods described herein, the subjecthas been identified or diagnosed as having a cancer. Some embodiments ofany of the methods described herein further include: obtaining themammalian cell from the subject. In some embodiments of any of themethods described herein, the contacting step is performed in vitro. Insome embodiments of any of the methods described herein, the contactingstep is performed in a mammal.

Also provided herein are single-chain chimeric antigen receptors thatinclude: an extracellular antigen-binding domain; a transmembranedomain; a hormone receptor ligand binding domain; a costimulatorydomain; and a immunoreceptor tyrosine-based activation motif (ITAM);where: the transmembrane domain and the hormone receptor ligand bindingdomain directly abut each other or are separated by 1 to 500 aminoacids; and the transmembrane domain and the hormone receptor ligandbinding domain are not both present in the same endogenous single-chainpolypeptide in a mammal. In some embodiments of any of the single-chainchimeric antigen receptors described herein, the extracellularantigen-binding domain is selected from the group of: a scFv, a (scFv)₂,a V_(H)H domain, and a V_(NAR) domain. In some embodiments of any of thesingle-chain chimeric antigen receptors described herein, theextracellular antigen-binding domain is a scFv.

In some embodiments of any of the single-chain chimeric antigenreceptors described herein, the extracellular antigen-binding domainbinds specifically to a single antigen. In some embodiments of any ofthe single-chain chimeric antigen receptors described herein, the singleantigen is a tumor antigen. In some embodiments of any of thesingle-chain chimeric antigen receptors described herein, the tumorantigen is selected from the group of: CD19, WT-1, CD22, L1-CAM, ROR-1,CD30, CD125, AFP, CEA, ETA, MAGE, and MUC16. In some embodiments of anyof the single-chain chimeric antigen receptors described herein, thetumor antigen is CD19. In some embodiments of any of the single-chainchimeric antigen receptors described herein, the extracellularantigen-binding domain binds specifically to two different antigens.

In some embodiments of any of the single chain chimeric antigenreceptors described herein, the transmembrane domain is a transmembranedomain from: α chain of a T cell receptor, β chain of the T cellreceptor, ζ chain of the T cell receptor, CD28, CD3ε, CD3δ, CD3γ, CD33,CD37, CD64, CD80, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD86, CD134,CD137, or CD154. In some embodiments of any of the single-chain chimericantigen receptors described herein, the hormone receptor ligand bindingdomain is a ligand binding domain from an estrogen receptor, aprogesterone receptor, or an androgen receptor. In some embodiments ofany of the single-chain chimeric antigen receptors described herein, thehormone receptor ligand binding domain is a ligand binding domain froman estrogen receptor.

In some embodiments of any of the single-chain chimeric antigenreceptors described herein, the costimulatory domain is a cytoplasmiccostimulatory domain from CD27, CD28, 4-1BB, OX40, CD30, CD40L, CD40,PD-1, PD-L1, ICOS, LFA-1, CD2, CD7, CD160, LIGHT, BTLA, TIM3, CD244,CD80, LAG3, NKG2C, and B7-H3. In some embodiments of any of thesingle-chain chimeric antigen receptors described herein, thecostimulatory domain is 4-1BB or CD28. In some embodiments of any of thesingle-chain chimeric antigen receptors described herein, the ITAMincludes a cytoplasmic signaling sequence from CD3ζ.

In some embodiments of any of the single-chain chimeric antigenreceptors described herein, the transmembrane domain and the hormonereceptor ligand binding domain directly abut each other. In someembodiments of any of the single-chain chimeric antigen receptorsdescribed herein, the transmembrane domain and the hormone receptorligand binding domain are separated by 1 to 500 amino acids (e.g., 1 to250 amino acids, or 1 to 50 amino acids).

Also provided herein are nucleic acids that include a nucleotidesequence encoding any of the single-chain chimeric antigen receptorsdescribed herein. Also provided herein are vectors that include anucleic acid that includes a nucleotide sequence encoding any of thesingle-chain chimeric antigen receptors described herein. Also providedare mammalian cells that include any of the vectors described herein.

Also provided are methods of inducing the membrane localization of asingle-chain chimeric antigen receptor in a mammalian cell that include:contacting a mammalian cell expressing any of the single-chain chimericantigen receptors described herein with an amount of a hormone or ahormone analogue sufficient to induce localization of the single-chainchimeric antigen-binding receptor to the extracellular side of theplasma membrane of the cell.

Also provided herein are methods of altering the membrane localizationof a single-chain chimeric antigen receptor in a mammalian cell thatinclude: (a) contacting a mammalian cell expressing any of thesingle-chain chimeric antigen receptors described herein with an amountof a hormone or a hormone analogue sufficient to induce localization ofthe single-chain chimeric antigen-binding receptor to the extracellularside of the plasma membrane of the cell; and (b) contacting themammalian cell with an amount of the hormone or the hormone analoguethat results in a reduced level of the single-chain chimeric antigenreceptor on the extracellular side of the plasma membrane of the cell.Some embodiments of these methods further include: (c) contacting themammalian cell with an amount of the hormone or the hormone analoguethat results in an increased level of single-chain chimeric antigenreceptor to the extracellular side of the plasma membrane of the cell ascompared to the level in (b).

Some embodiments of any of the methods described herein further include:introducing a nucleic acid encoding the single-chain chimeric antigenreceptor into a mammalian cell to generate the mammalian cell expressingthe single-chain chimeric antigen receptor. In some embodiments of anyof the methods described herein, the mammalian cell is a T cell. In someembodiments of any of the methods described herein, the T cell isselected from the group of: a CD8+ T cell, a CD4+ T cell, a memory Tcell, a Treg cell, and a natural killer T cell.

In some embodiments of any of the methods described herein, themammalian cell is a mammalian cell previously obtained from a subject.In some embodiments of any of the methods described herein, the subjecthas been identified or diagnosed as having a cancer. Some embodiments ofany of the methods described herein further include: obtaining themammalian cell from the subject. In some embodiments of any of themethods described herein, the contacting step is performed in vitro. Insome embodiments of any of the methods described herein, the contactingstep is performed in a mammal. In some embodiments of any of the methodsdescribed herein, the mammal has a cancer. In some embodiments of any ofthe methods described herein, the contacting step results in thetreatment of the cancer in the mammal.

Also provided herein are multi-chain chimeric antigen receptors thatinclude at least one first polypeptide including: an extracellularantigen-binding domain; a transmembrane domain; and a hormone receptorligand binding domain; where: the transmembrane domain and the hormonereceptor ligand binding domain directly abut each other or are separatedby 1 to 500 amino acids; and the transmembrane domain and the hormonereceptor ligand binding domain are not both present in the sameendogenous single-chain polypeptide in a mammal. In some embodiments ofany of the multi-chain chimeric antigen receptors described herein, theextracellular antigen-binding domain is selected from the groupconsisting of: a scFv, a (scFv)₂, a V_(H)H domain, and a V_(NAR) domain.In some embodiments of any of the multi-chain chimeric antigen receptorsdescribed herein, the extracellular antigen-binding domain is a scFv.

In some embodiments of any of the multi-chain chimeric antigen receptorsdescribed herein, the extracellular antigen-binding domain bindsspecifically to a single antigen. In some embodiments of any of themulti-chain chimeric antigen receptors described herein, the singleantigen is a tumor antigen. In some embodiments of any of themulti-chain chimeric antigen receptors described herein, the tumorantigen is selected from the group of: CD19, WT-1, CD22, L1-CAM, ROR-1,CD30, CD125, AFP, CEA, ETA, MAGE, and MUC16. In some embodiments of anyof the multi-chain chimeric antigen receptors described herein, thetumor antigen is CD19. In some embodiments of any of the multi-chainchimeric antigen receptors described herein, the extracellularantigen-binding domain binds specifically to two different antigens.

In some embodiments of any of the multi-chain chimeric antigen receptorsdescribed herein, the transmembrane domain is a transmembrane domainfrom: α chain of a T cell receptor, β chain of the T cell receptor, ζchain of the T cell receptor, CD28, CD3ε, CD3δ, CD3γ, CD33, CD37, CD64,CD80, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD86, CD134, CD137, orCD154. In some embodiments of any of the multi-chain chimeric antigenreceptors described herein, the hormone receptor ligand binding domainis a ligand binding domain from an estrogen receptor, a progesteronereceptor, or an androgen receptor. In some embodiments of any of themulti-chain chimeric antigen receptors described herein, the hormonereceptor ligand binding domain is a ligand binding domain from anestrogen receptor.

In some embodiments of any of the multi-chain chimeric antigen receptorsdescribed herein, the transmembrane domain and the hormone receptorligand binding domain directly abut each other. In some embodiments ofany of the multi-chain chimeric antigen receptors described herein, thetransmembrane domain and the hormone receptor ligand binding domain areseparated by 1 to 500 amino acids (e.g., 1 to 250 amino acids, or 1 to50 amino acids).

In some embodiments of any of the multi-chain chimeric antigen receptorsdescribed herein, the at least one polypeptide further includes acostimulatory domain. In some embodiments of any of the multi-chainchimeric antigen receptors described herein, the costimulatory domain isa cytoplasmic costimulatory domain from CD27, CD28, 4-1BB, OX40, CD30,CD40L, CD40, PD-1, PD-L1, ICOS, LFA-1, CD2, CD7, CD160, LIGHT, BTLA,TIM3, CD244, CD80, LAG3, NKG2C, or B7-H3. In some embodiments of any ofthe multi-chain chimeric antigen receptors described herein, thecostimulatory domain is 4-1BB or CD28.

Also provided herein are methods of inducing membrane localization of amulti-chain chimeric antigen receptor in a mammalian cell that include:contacting a mammalian cell expressing any of the multi-chain chimericantigen receptors described herein with an amount of a hormone or ahormone analogue sufficient to induce localization of the multi-chainchimeric antigen-binding receptor to the extracellular side of theplasma membrane of the cell.

Also provided herein are methods of altering the membrane localizationof a multi-chain chimeric antigen receptor in a mammalian cell thatinclude: (a) contacting a mammalian cell expressing any of themulti-chain chimeric antigen receptors described herein with an amountof a hormone or a hormone analogue sufficient to induce localization ofthe multi-chain chimeric antigen-binding receptor to the extracellularside of the plasma membrane of the cell; and (b) contacting themammalian cell with an amount of the hormone or the hormone analoguewith an amount of the hormone or the hormone analogue that results in adecreased level of the multi-chain chimeric antigen-binding receptor onthe extracellular side of the plasma membrane of the cell. Someembodiments of any of the methods described herein further include: (c)contacting the mammalian cell with an amount of the hormone or thehormone analogue that results in an increase in the level of themulti-chain chimeric antigen-binding receptor on the extracellular sideof the plasma membrane of the cell. Some embodiments of any of themethods described herein further include: introducing a nucleic acidencoding the single-chain chimeric antigen receptor into a mammaliancell to generate the mammalian cell expressing the single-chain chimericantigen receptor. In some embodiments of any of the methods describedherein, the mammalian cell is a T cell. In some embodiments of any ofthe methods described herein, the T cell is selected from the group of:a CD8+ T cell, a CD4+ T cell, a memory T cell, a Treg cell, and anatural killer T cell.

In some embodiments of any of the methods described herein, themammalian cell is a mammalian cell previously obtained from a subject.In some embodiments of any of the methods described herein, the subjecthas been identified or diagnosed as having a cancer. Some embodiments ofany of the methods described herein further include: obtaining themammalian cell from the subject. In some embodiments of any of themethods described herein, the contacting step is performed in vitro. Insome embodiments of any of the methods described herein, the contactingstep is performed in a mammal. In some embodiments of any of the methodsdescribed herein, the mammal has a cancer. In some embodiments of any ofthe methods described herein, the contacting step results in thetreatment of the cancer in the mammal.

Also provided herein are methods of treating a cancer in a subject(e.g., a human) that include: (a) administering to the subject amammalian cell expressing any of the single chain chimeric antigenreceptors described herein or any of the multi-chain chimeric antigenreceptors described herein, where the antigen binding domain bindsspecifically to an antigen expressed by the cancer in the subject; (b)administering to the subject an amount of a hormone or hormonesufficient to induce localization of the single-chain or multi-chainchimeric antigen receptor to the extracellular side of the plasmamembrane of the cell; (c) determining the presence or severity of one ormore symptoms associated with toxicity, the level of one or morecytokines associated with a cytokine storm, or both, in the subjectfollowing (b); (d) comparing the determined presence or severity of oneor more symptoms associated with toxicity, the determined level of oneor more cytokines associated with the cytokine storm, or both, to acontrol(s); and (e) administering to the subject an amount of thehormone or hormone analogue sufficient to reduce the level of thesingle-chain or multi-chain chimeric antigen receptor on theextracellular side of the plasma membrane of the cell, such that thepresence or severity of one or more symptoms associated with toxicity,the level of one or more cytokines associated with a cytokine storm, orboth, is reduced in the subject after (e).

In some embodiments of any of the methods described herein, the amountof the hormone or hormone analogue administered to the subject in step(e) is reduced by at least 25% as compared to the amount of the hormoneor hormone analogue administered to the subject in step (b). In someembodiments of any of the methods described herein, the amount of thehormone or hormone analogue administered to the subject in step (e) isreduced by at least 50% as compared to the amount of the hormone orhormone analogue administered to the subject in step (b). In someembodiments of any of the methods described herein, the amount of thehormone or hormone analogue administered to the subject in step (e) isreduced by at least 75% as compared to the level of the hormone orhormone analogue administered to the subject in step (b). In someembodiments of any of the methods described herein, the amount of thehormone or hormone analogue administered to the subject in step (e) isreduced by 100% as compared to the level of the hormone or hormoneanalogue administered to the subject in step (b).

In some embodiments of any of the methods described herein, the one ormore symptoms associated with toxicity are selected from the groupconsisting of: high fever, swelling, chills, hypotension, tachycardia,asthenia, headache, rash, scratchy throat, dyspnea, redness, extremefatigue, nausea, and cerebral edema. In some embodiments of thesemethods, the one or more cytokines associated with a cytokine storm areselected from the group consisting of: include tumor necrosisfactor-alpha, IFNγ, IL-10, IL-1β, IL-2, IL-6, IL-8, and IL-10,granulocyte macrophage-colony-stimulating factor (GM-CSF), and IL-5.

Also provided herein are single-chain chimeric polypeptides thatinclude: an extracellular hormone receptor ligand binding domain; and atransmembrane domain, where: the hormone receptor ligand binding domainand the extracellular transmembrane domain directly abut each other orare separated by 1 to 700 amino acids; and the transmembrane domain andthe extracellular hormone receptor ligand binding domain are not bothpresent in a single endogenous single-chain polypeptide in a mammal. Insome embodiments of any of the single-chain chimeric polypeptidesdescribed herein, the transmembrane domain is a transmembrane domainfrom: α chain of a T cell receptor, β chain of the T cell receptor, ζchain of the T cell receptor, CD28, CD3ε, CD3δ, CD3γ, CD33, CD37, CD64,CD80, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD86, CD134, CD137, orCD154. In some embodiments of any of the single chain chimericpolypeptides described herein, the extracellular hormone receptor ligandbinding domain is a ligand binding domain from an estrogen receptor, aprogesterone receptor, or an androgen receptor. In some embodiments ofany of the single-chain chimeric polypeptides described herein, theextracellular hormone receptor ligand binding domain is a ligand bindingdomain from an estrogen receptor. In some embodiments of any of thesingle-chain chimeric polypeptides described herein, the extracellularhormone receptor ligand binding domain is a ligand binding domain from ahuman estrogen receptor. In some embodiments of any of the single-chainchimeric polypeptides described herein, the ligand binding domain of thehuman estrogen receptor has a wildtype sequence, except that itcomprises one or both of a substitution at amino acid position 521 and asubstitution at amino acid position 537, each numbered relative to thefull-length wildtype sequence of human hormone receptor.

In some embodiments of any of the single-chain chimeric polypeptidedescribed herein, the extracellular hormone receptor ligand bindingdomain and the transmembrane domain directly abut each other. In someembodiments of any of the single-chain chimeric polypeptides describedherein, the extracellular hormone receptor ligand binding domain and thetransmembrane domain are separated by 1 to 700 amino acids (e.g., 1 to500 amino acids, 1 to 250 amino acids, 1 to 100 amino acids, 1 to 50amino acids, or 1 to 25 amino acids).

In some embodiments of any of the single-chain chimeric polypeptidesdescribed herein, when going in the N-terminal to C-terminal direction,the single-chain chimeric polypeptide includes the extracellularantigen-binding domain, the extracellular hormone receptor ligandbinding domain, and the transmembrane domain. In some embodiments of anyof the single-chain chimeric polypeptides described herein, when goingin the C-terminal to the N-terminal direction, the single-chain chimericpolypeptide includes the extracellular antigen-binding domain, theextracellular hormone receptor ligand binding domain, and thetransmembrane domain. In some embodiments of any of the single-chainchimeric polypeptides described herein, when going in the N-terminal tothe C-terminal direction, the single-chain chimeric polypeptide includesthe extracellular hormone receptor ligand binding domain, theextracellular antigen-binding domain, and the transmembrane domain. Insome embodiments of any of the single-chain chimeric polypeptidesdescribed herein, when going in the C-terminal to the N-terminaldirection, the single-chain chimeric polypeptide includes theextracellular hormone receptor ligand binding domain, the extracellularantigen-binding domain, and the transmembrane domain.

Also provided herein are nucleic acids that include a nucleotidesequence encoding any of the single-chain chimeric polypeptidesdescribed herein. Also provided herein are vectors that include anucleic acid that includes a nucleotide sequence encoding any of thesingle-chain chimeric polypeptides described herein. Also providedherein are mammalian cells that include any of the vectors describedherein. In some embodiments of any of the mammalian cells describedherein, the mammalian cell is a T cell. In some embodiments of any ofthe mammalian cells described herein, the T cell is selected from thegroup of: a CD8⁺ T cell, a CD4⁺ T cell, a memory T cell, a Treg cell,and natural killer T cell.

Also provided herein are methods of inducing the plasma membranelocalization of a single-chain chimeric polypeptide in a mammalian cellthat include: (a) contacting a mammalian cell expressing any of thesingle-chain chimeric polypeptides described herein with an amount of ahormone or a hormone analogue sufficient to induce localization of thesingle-chain chimeric polypeptide to the extracellular side of theplasma membrane of the cell.

Also provided herein are methods of reversibly altering the plasmamembrane localization of a single-chain chimeric polypeptide in amammalian cell that include: (a) contacting a mammalian cell expressingany of the single-chain chimeric polypeptides described herein with anamount of a hormone or a hormone analogue sufficient to inducelocalization of the single-chain chimeric polypeptide to theextracellular side of the plasma membrane of the cell; and (b)contacting the mammalian cell with a reduced amount of the hormone orthe hormone analogue that results in a decreased level of thesingle-chain chimeric polypeptide on the extracellular side of theplasma membrane of the mammalian cell. Some embodiments of any of themethods described herein further include: (c) contacting the mammaliancell with an increased amount of the hormone or the hormone analoguethat results in an increased level of the single-chain chimericpolypeptide on the extracellular side of the plasma membrane of the cellas compared to the level of in step (b). Some embodiments of any of themethods described herein further include, before step (a), introducing anucleic acid encoding the single-chain chimeric polypeptide into amammalian cell to generate the mammalian cell expressing thesingle-chain chimeric polypeptide.

In some embodiments of any of the methods described herein, themammalian cell is a T cell. In some embodiments of any of the methodsdescribed herein, the T cell is selected from the group of: a CD8⁺ Tcell, a CD4⁺ T cell, a memory T cell, a Treg cell, and natural killer Tcell. In some embodiments of any of the methods described herein, themammalian cell is a mammalian cell previously obtained from a subject.In some embodiments of any of the methods described herein, the subjecthas been identified or diagnosed as having a cancer. Some embodiments ofany of the methods described herein further include: obtaining themammalian cell from the subject.

In some embodiments of any of the methods described herein, thecontacting step(s) is performed in vitro. In some embodiments of any ofthe methods described herein, the contacting step(s) is performed in amammal.

Also provided herein are single-chain chimeric antigen receptors thatinclude: an extracellular antigen-binding domain; an extracellularhormone receptor ligand binding domain; a transmembrane domain; anintracellular costimulatory domain; and an intracellular immunoreceptortyrosine-based activation motif (ITAM); where: the transmembrane domainand the extracellular hormone receptor ligand binding domain directlyabut each other or are separated by 1 to 700 amino acids; and thetransmembrane domain and the extracellular hormone receptor ligandbinding domain are not both present in a single endogenous single-chainpolypeptide in a mammal. In some embodiments of any of the single-chainchimeric antigen receptors described herein, the extracellularantigen-binding domain is selected from the group consisting of: a scFv,a (scFv)₂, a V_(H)H domain, and a V_(NAR) domain. In some embodiments ofany of the single-chain chimeric antigen receptors described herein, theextracellular antigen-binding domain is a scFv.

In some embodiments of any of the single-chain chimeric antigenreceptors described herein, the extracellular antigen-binding domainbinds specifically to a single antigen. In some embodiments of any ofthe single-chain chimeric antigen receptors, the single antigen is atumor antigen. In some embodiments of any of the single-chain chimericantigen receptors described herein, the tumor antigen is selected fromthe group of: CD19, WT-1, CD22, L1-CAM, ROR-1, CD30, CD125, AFP, CEA,ETA, MAGE, and MUC16. In some embodiments of any of the single-chainchimeric antigen receptors described herein, the tumor antigen is CD19.In some embodiments of any of the single-chain chimeric antigenreceptors described herein, the extracellular antigen-binding domainbinds specifically to two different antigens. In some embodiments of anyof the single-chain chimeric antigen receptors described herein, atleast one of the two different antigens is a tumor antigen. In someembodiments of any of the single-chain chimeric antigen receptorsdescribed herein, the tumor antigen is selected from the group of: CD19,WT-1, CD22, L1-CAM, ROR-1, CD30, CD125, AFP, CEA, ETA, MAGE, and MUC16.In some embodiments of any of the single-chain chimeric antigenreceptors described herein, the tumor antigen is CD19.

In some embodiments of any of the single-chain chimeric antigenreceptors described herein, the transmembrane domain is a transmembranedomain from: α chain of a T cell receptor, β chain of the T cellreceptor, ζ chain of the T cell receptor, CD28, CD3ε, CD3δ, CD3γ, CD33,CD37, CD64, CD80, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD86, CD134,CD137, or CD154. In some embodiments of any of the single-chain chimericantigen receptors described herein, the extracellular hormone receptorligand binding domain is a ligand binding domain from an estrogenreceptor, a progesterone receptor, or an androgen receptor. In someembodiments of any of the single-chain chimeric antigen receptorsdescribed herein, the extracellular hormone receptor ligand bindingdomain is a ligand binding domain from an estrogen receptor. In someembodiments of any of the single-chain chimeric antigen receptorsdescribed herein, the extracellular hormone receptor ligand bindingdomain is a ligand binding domain from a human estrogen receptor. Insome embodiments of any of the single-chain chimeric antigen receptorsdescribed herein, the ligand binding domain of the human estrogenreceptor has a wildtype sequence, except that it comprises one or bothof a substitution at amino acid position 521 and a substitution at aminoacid position 537, each numbered relative to the full-length wildtypesequence of human hormone receptor.

In some embodiments of any of the single-chain chimeric antigenreceptors described herein, the intracellular costimulatory domain is acytoplasmic costimulatory domain from CD27, CD28, 4-1BB, OX40, CD30,CD40L, CD40, PD-1, PD-L1, ICOS, LFA-1, CD2, CD7, CD160, LIGHT, BTLA,TIM3, CD244, CD80, LAG3, NKG2C, or B7-H3. In some embodiments of any ofthe single-chain chimeric antigen receptors described herein, theintracellular costimulatory domain is the cytoplasmic costimulatorydomain from 4-1BB or CD28. In some embodiments of any of thesingle-chain chimeric antigen receptors described herein, theintracellular ITAM comprises a cytoplasmic signaling sequence from CD3ζ.

In some embodiments of any of the single-chain chimeric antigenreceptors described herein, the extracellular hormone receptor ligandbinding domain and the transmembrane domain directly abut each other. Insome embodiments of any of the single-chain chimeric antigen receptorsdescribed herein, the extracellular hormone receptor ligand bindingdomain and the transmembrane domain are separated by 1 to 700 aminoacids (e.g., 1 to 500 amino acids, 1 to 250 amino acids, 1 to 100 aminoacids, 1 to 50 amino acids, or 1 to 25 amino acids).

In some embodiments of any of the single-chain chimeric antigenreceptors described herein, when going in the N-terminal to C-terminaldirection, the single-chain chimeric antigen receptor includes theextracellular antigen-binding domain, the extracellular hormone receptorligand binding domain, the transmembrane domain, the intracellularcostimulatory domain, and the ITAM. In some embodiments of any of thesingle-chain chimeric antigen receptors described herein, when going inthe C-terminal to the N-terminal direction, the single-chain chimericantigen receptor includes the extracellular antigen-binding domain, theextracellular hormone receptor ligand binding domain, the transmembranedomain, the intracellular costimulatory domain, and the ITAM. In someembodiments of any of the single-chain chimeric antigen receptorsdescribed herein, when going in the N-terminal to the C-terminaldirection, the single-chain chimeric antigen receptor comprises theextracellular hormone receptor ligand binding domain, the extracellularantigen-binding domain, the transmembrane domain, the intracellularcostimulatory domain, and the ITAM. In some embodiments of any of thesingle-chain chimeric antigen receptors described herein, when going inthe C-terminal to the N-terminal direction, the single-chain chimericpolypeptide includes the extracellular hormone receptor ligand bindingdomain, the extracellular antigen-binding domain, the transmembranedomain, the intracellular costimulatory domain, and the ITAM.

Also provided herein are nucleic acids that include a nucleotidesequence encoding any of the single-chain chimeric antigen receptorsdescribed herein. Also provided herein are vectors that include anucleic acid that includes a nucleotide sequence encoding any of thesingle-chain chimeric antigen receptors described herein. Also providedare mammalian cells that include any of the vectors described herein. Insome embodiments of any of the mammalian cells described herein, themammalian cell is a T cell. In some embodiments of any of the mammaliancells described herein, the T cell is selected from the group of: a CD8⁺T cell, a CD4⁺ T cell, a memory T cell, a Treg cell, and natural killerT cell.

Also provided are methods of inducing the plasma membrane localizationof a single-chain chimeric antigen receptor in a mammalian cell thatinclude: (a) contacting a mammalian cell expressing any of thesingle-chain chimeric antigen receptors described herein with an amountof a hormone or a hormone analogue sufficient to induce localization ofthe single-chain chimeric antigen-binding receptor to the extracellularside of the plasma membrane of the cell.

Also provided herein are methods of altering the plasma membranelocalization of a single-chain chimeric antigen receptor in a mammaliancell that include: (a) contacting a mammalian cell expressing any of thesingle-chain chimeric antigen receptors described herein with an amountof a hormone or a hormone analogue sufficient to induce localization ofthe single-chain chimeric antigen-binding receptor to the extracellularside of the plasma membrane of the cell; and (b) contacting themammalian cell with an amount of the hormone or the hormone analoguethat results in a reduced level of the single-chain chimeric antigenreceptor on the extracellular side of the plasma membrane of the cell.Some embodiments of any of the methods described herein further include:(c) contacting the mammalian cell with an amount of the hormone or thehormone analogue that results in an increased level of single-chainchimeric antigen receptor to the extracellular side of the plasmamembrane of the cell as compared to the level in (b). Some embodimentsof any of the methods described herein further include before step (a):introducing a nucleic acid encoding the single-chain chimeric antigenreceptor into a mammalian cell to generate the mammalian cell expressingthe single-chain chimeric antigen receptor.

In some embodiments of any of the methods described herein, themammalian cell is a T cell. In some embodiments of any of the methodsdescribed herein, the T cell is selected from the group of: a CD8⁺ Tcell, a CD4⁺ T cell, a memory T cell, a Treg cell, and a natural killerT cell. In some embodiments of any of the methods described herein, themammalian cell is a mammalian cell previously obtained from a subject.In some embodiments of any of the methods described herein, the subjecthas been identified or diagnosed as having a cancer. Some embodiments ofany of the methods described herein further include: obtaining themammalian cell from the subject. In some embodiments of any of themethods described herein, the contacting step(s) is performed in vitro.In some embodiments of any of the methods described herein, thecontacting step(s) is performed in a mammal. In some embodiments of anyof the methods described herein, the mammal has a cancer. In someembodiments of any of the methods described herein, the contacting stepresults in the treatment of the cancer in the mammal.

Also provided herein are multi-chain chimeric antigen receptors thatinclude at least one first polypeptide including: an extracellularantigen-binding domain; an extracellular hormone receptor ligand bindingdomain; and a transmembrane domain; where: the transmembrane domain andthe extracellular hormone receptor ligand binding domain directly abuteach other or are separated by 1 to 700 amino acids; and thetransmembrane domain and the extracellular hormone receptor ligandbinding domain are not both present in a single endogenous single-chainpolypeptide in a mammal. In some embodiments of any of the multi-chainchimeric antigen receptors described herein, the extracellularantigen-binding domain is selected from the group consisting of: a scFv,a (scFv)₂, a V_(H)H domain, and a V_(NAR) domain. In some embodiments ofany of the multi-chain chimeric antigen receptors described herein, theextracellular antigen-binding domain is a scFv.

In some embodiments of any of the multi-chain chimeric antigen receptorsdescribed herein, the extracellular antigen-binding domain bindsspecifically to a single antigen. In some embodiments of any of themulti-chain chimeric antigen receptors described herein, the singleantigen is a tumor antigen. In some embodiments of any of themulti-chain chimeric antigen receptors described herein, the tumorantigen is selected from the group of: CD19, WT-1, CD22, L1-CAM, ROR-1,CD30, CD125, AFP, CEA, ETA, MAGE, and MUC16. In some embodiments of anyof the multi-chain chimeric antigen receptors described herein, thetumor antigen is CD19. In some embodiments of any of the multi-chainchimeric antigen receptors described herein, the extracellularantigen-binding domain binds specifically to two different antigens. Insome embodiments of any of the multi-chain chimeric antigen receptorsdescribed herein at least one of the two different antigens is a tumorantigen. In some embodiments of any of the multi-chain chimeric antigenreceptors described herein, the tumor antigen is selected from the groupof: CD19, WT-1, CD22, L1-CAM, ROR-1, CD30, CD125, AFP, CEA, ETA, MAGE,and MUC16. In some embodiments of any of the multi-chain chimericantigen receptors described herein, the tumor antigen is CD19.

In some embodiments of any of the multi-chain chimeric antigen receptorsdescribed herein, the transmembrane domain is a transmembrane domainfrom: α chain of a T cell receptor, β chain of the T cell receptor, ζchain of the T cell receptor, CD28, CD3ε, CD3δ, CD3γ, CD33, CD37, CD64,CD80, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD86, CD134, CD137, orCD154. In some embodiments of any of the multi-chain chimeric antigenreceptors described herein, the hormone receptor ligand binding domainis a ligand binding domain from an estrogen receptor, a progesteronereceptor, or an androgen receptor. In some embodiments of any of themulti-chain chimeric antigen receptors described herein, the hormonereceptor ligand binding domain is a ligand binding domain from anestrogen receptor. In some embodiments of any of the multi-chainchimeric antigen receptors described herein, the extracellular hormonereceptor ligand binding domain is a ligand binding domain from a humanestrogen receptor. In some embodiments of any of the multi-chainchimeric antigen receptors described herein, the ligand binding domainof the human estrogen receptor has a wildtype sequence, except that itincludes one or both of a substitution at amino acid position 521 and asubstitution at amino acid position 537, each numbered relative to thefull-length wildtype sequence of human hormone receptor.

In some embodiments of any of the multi-chain chimeric antigen receptorsdescribed herein, the extracellular hormone receptor ligand bindingdomain and the transmembrane domain directly abut each other. In someembodiments of any of the multi-chain chimeric antigen receptorsdescribed herein, the extracellular hormone receptor ligand bindingdomain and the transmembrane domain are separated by 1 to 700 aminoacids (e.g., 1 to 500 amino acids, 1 to 250 amino acids, 1 to 100 aminoacids, 1 to 50 amino acids, or 1 to 25 amino acids).

In some embodiments of any of the multi-chain chimeric antigen receptorsdescribed herein, the at least one polypeptide further includes anintracellular costimulatory domain. In some embodiments of any of themulti-chain chimeric antigen receptors described herein, theintracellular costimulatory domain is a cytoplasmic costimulatory domainfrom CD27, CD28, 4-1BB, OX40, CD30, CD40L, CD40, PD-1, PD-L1, ICOS,LFA-1, CD2, CD7, CD160, LIGHT, BTLA, TIM3, CD244, CD80, LAG3, NKG2C, orB7-H3. In some embodiments of any of the multi-chain chimeric antigenreceptors described herein, the the intracellular costimulatory domainis the cytoplasmic costimulatory domain from 4-1BB or CD28.

In some embodiments of any of the multi-chain chimeric antigen receptorsdescribed herein, when going in the N-terminal to C-terminal direction,the at least one first polypeptide includes the extracellularantigen-binding domain, the extracellular hormone receptor ligandbinding domain, and the transmembrane domain. In some embodiments of anyof the multi-chain chimeric antigen receptors described herein, whengoing in the C-terminal to N-terminal direction, the at least one firstpolypeptide includes the extracellular antigen-binding domain, theextracellular hormone receptor ligand binding domain, and thetransmembrane domain. In some embodiments of any of the multi-chainchimeric antigen receptors described herein, when going in theN-terminal to C-terminal direction, the at least one first polypeptideincludes the extracellular hormone receptor ligand binding domain, theextracellular antigen-binding domain, and the transmembrane domain. Insome embodiments of any of the multi-chain chimeric antigen receptorsdescribed herein, when going in the C-terminal to N-terminal direction,the at least one first polypeptide includes the extracellular hormonereceptor ligand binding domain, the extracellular antigen-bindingdomain, and the transmembrane domain.

Also provided herein are nucleic acids that encode any of themulti-chain chimeric antigen receptors described herein. Also providedherein are sets of nucleic acids that together encode any of themulti-chain chimeric antigen receptors described herein. Also providedherein are mammalian cells that include a nucleic acid that encodes anyof the multi-chain chimeric antigen receptors described herein. Alsoprovided herein are mammalian cells that include a set of nucleic acidsthat together encode any of the multi-chain chimeric antigen receptorsdescribed herein. In some embodiments of any of the mammalian cellsdescribed herein, the mammalian cell is a T cell. In some embodiments ofany of the mammalian cells described herein, the T cell is selected fromthe group consisting of: a CD8⁺ T cell, a CD4⁺ T cell, a memory T cell,a Treg cell, and natural killer T cell.

Also provided herein are methods of inducing the plasma membranelocalization of a multi-chain chimeric antigen receptor in a mammaliancell that include: (a) contacting a mammalian cell expressing any of themulti-chain chimeric antigen receptors described herein with an amountof a hormone or a hormone analogue sufficient to induce localization ofthe multi-chain chimeric antigen-binding receptor to the extracellularside of the plasma membrane of the cell.

Also provided herein are methods of altering the plasma membranelocalization of a multi-chain chimeric antigen receptor in a mammaliancell that include: (a) contacting a mammalian cell expressing any of themulti-chain chimeric antigen receptors described herein with an amountof a hormone or a hormone analogue sufficient to induce localization ofthe multi-chain chimeric antigen-binding receptor to the extracellularside of the plasma membrane of the cell; and (b) contacting themammalian cell with an amount of the hormone or the hormone analoguewith an amount of the hormone or the hormone analogue that results in adecreased level of the multi-chain chimeric antigen-binding receptor onthe extracellular side of the plasma membrane of the cell. Someembodiments of any of the methods described herein further include: (c)contacting the mammalian cell with an amount of the hormone or thehormone analogue that results in an increase in the level of themulti-chain chimeric antigen-binding receptor on the extracellular sideof the plasma membrane of the cell. Some embodiments of any of themethods described herein further include before step (a): introducing anucleic acid encoding the multi-chain chimeric antigen receptor or a setof nucleic acids that together encode the multi-chain chimeric receptor,into a mammalian cell to generate the mammalian cell expressing themulti-chain chimeric antigen receptor. In some embodiments of any of themethods described herein, the mammalian cell is a T cell. In someembodiments of any of the methods described herein, the T cell isselected from the group of: a CD8⁺ T cell, a CD4⁺ T cell, a memory Tcell, a Treg cell, and a natural killer T cell. In some embodiments ofany of the methods described herein, the mammalian cell is a mammaliancell previously obtained from a subject. In some embodiments of any ofthe methods described herein, the subject has been identified ordiagnosed as having a cancer. Some embodiments of any of the methodsdescribed herein further include obtaining the mammalian cell from thesubject. In some embodiments of any of the methods described herein, thecontacting step is performed in vitro. In some embodiments of any of themethods described herein, the contacting step is performed in a mammal.In some embodiments of any of the methods described herein, the mammalhas a cancer. In some embodiments of any of the methods describedherein, the contacting step results in the treatment of the cancer inthe mammal.

Also provided herein are methods of treating a cancer in a subject thatinclude: (a) administering to the subject a mammalian cell expressingany of the single-chain chimeric antigen receptors described herein orany of the multi-chain chimeric antigen receptors described herein,where the antigen binding domain binds specifically to an antigenexpressed by the cancer in the subject; (b) administering to the subjectan amount of a hormone or hormone sufficient to induce localization ofthe single-chain or multi-chain chimeric antigen receptor to theextracellular side of the plasma membrane of the cell; (c) determiningthe presence or severity of one or more symptoms associated withtoxicity or the level of one or more cytokines associated with acytokine storm, or both, in the subject following (b); (d) comparing thedetermined presence or severity of one or more symptoms associated withtoxicity or the determined level of one or more cytokines associatedwith the cytokine storm, or both, to a control(s); and (e) administeringto the subject an amount of the hormone or hormone analogue sufficientto reduce the level of the single-chain or multi-chain chimeric antigenreceptor on the extracellular side of the plasma membrane of the cell,such that the presence or severity of one or more symptoms associatedwith toxicity or the level of one or more cytokines associated with acytokine storm, or both, is reduced in the subject after (e).

In some embodiments of any of the methods described herein, the amountof the hormone or hormone analogue administered to the subject in step(e) is reduced by at least 25% as compared to the amount of the hormoneor hormone analogue administered to the subject in step (b). In someembodiments of any of the methods described herein, the amount of thehormone or hormone analogue administered to the subject in step (e) isreduced by at least 50% as compared to the amount of the hormone orhormone analogue administered to the subject in step (b). In someembodiments of any of the methods described herein, the amount of thehormone or hormone analogue administered to the subject in step (e) isreduced by at least 75% as compared to the level of the hormone orhormone analogue administered to the subject in step (b). In someembodiments of any of the methods described herein, the amount of thehormone or hormone analogue administered to the subject in step (e) isreduced by 100% as compared to the level of the hormone or hormoneanalogue administered to the subject in step (b).

In some embodiments of any of the methods described herein, the one ormore symptoms associated with toxicity are selected from the group of:high fever, swelling, chills, hypotension, tachycardia, asthenia,headache, rash, scratchy throat, dyspnea, redness, extreme fatigue,nausea, and cerebral edema. In some embodiments of any of the methodsdescribed herein, the one or more cytokines associated with a cytokinestorm are selected from the group of: include tumor necrosisfactor-alpha, IFNγ, IL-10, IL-1β, IL-2, IL-6, IL-8, and IL-10,granulocyte macrophage-colony-stimulating factor (GM-CSF), and IL-5.

The use of the term “a” before a noun is meant “one or more” of theparticular noun. For example, the phrase “a mammalian cell” means “oneor more mammalian cell.”

The term “single-chain chimeric polypeptide” means a single-chainpolypeptide that includes a first contiguous amino acid sequence (e.g.,a first domain) and a second contiguous amino acid sequence (e.g., asecond domain), where the first contiguous amino acid sequence and thesecond contiguous amino acid sequence are not present in the sameendogenous single-chain polypeptide in a mammal. In some examples, thefirst contiguous amino acid sequence and the second contiguous aminoacid sequence directly abut each other in the single-chain chimericpolypeptide. In some examples, the first contiguous amino acid sequenceand the second contiguous amino acid sequence are separated by one ormore intervening amino acids (e.g., one or more domains). The term“single-chain chimeric polypeptide” is not meant to limit in any way therelative positioning of the first contiguous amino acid sequence and thesecond contiguous sequence in the single-chain polypeptide. Non-limitingexamples of single-chain chimeric polypeptides (e.g., single-chainchimeric antigen receptors) are described herein. Additional examples ofsingle-chain chimeric polypeptides (e.g., single-chain chimeric antigenreceptors) are known in the art.

The terms “chimeric antigen receptor” and “CAR” are used interchangeablyherein, and refer to artificial multi-module molecules capable oftriggering or inhibiting the activation of an immune cell whichgenerally but not exclusively comprise an extracellular domain (e.g., aligand/antigen binding domain), a transmembrane domain and one or moreintracellular signaling domains. CAR molecules and derivatives thereof(e.g., CAR variants) are described, e.g., in PCT Application No.US2014/016527; Fedorov et al. Sci Transl Med (2013); 5(215):215ra172;Glienke et al. Front Pharmacol (2015) 6:21; Kakarla & Gottschalk 52Cancer J (2014) 20(2):151-5; Riddell et al. Cancer J (2014) 20(2):141-4;Pegram et al. Cancer J (2014) 20(2):127-33; Cheadle et al. Immunol Rev(2014) 257(1):91-106; Barrett et al. Annu Rev Med (2014) 65:333-47;Sadelain et al. Cancer Discov (2013) 3(4):388-98; Cartellieri et al., JBiomed Biotechnol (2010) 956304; the disclosures of which areincorporated herein by reference in their entirety.

The term “multi-chain chimeric polypeptide” means a polypeptideincluding two or more single-chain polypeptides, where at least one ofthe two or more single-chain polypeptides is a single-chain chimericpolypeptide as defined and described herein. Non-limiting examples ofmulti-chain chimeric polypeptides (e.g., multi-chain chimeric antigenreceptors) are described herein. Additional examples of multi-chainchimeric polypeptides (e.g., multi-chain chimeric antigen receptors) areknown in the art.

The term “transmembrane domain” means a domain of a polypeptide thatincludes at least one contiguous amino acid sequence that traverses alipid bilayer when present in the corresponding endogenous polypeptidewhen expressed in a mammalian cell. For example, a transmembrane domaincan include one, two, three, four, five, six, seven, eight, nine, or tencontiguous amino acid sequences that each traverse a lipid bilayer whenpresent in the corresponding endogenous polypeptide when expressed in amammalian cell. As is known in the art, a transmembrane domain can,e.g., include at least one (e.g., two, three, four, five, six, seven,eight, nine, or ten) contiguous amino acid sequence (that traverses alipid bilayer when present in the corresponding endogenous polypeptidewhen expressed in a mammalian cell) that has α-helical secondarystructure in the lipid bilayer. In some embodiments, a transmembranedomain can include two or more contiguous amino acid sequences (thateach traverse a lipid bilayer when present in the correspondingendogenous polypeptide when expressed in a mammalian cell) that form aβ-barrel secondary structure in the lipid bilayer. Non-limiting examplesof transmembrane domains are described herein. Additional examples oftransmembrane domains are known in the art.

The phrase “extracellular side of the plasma membrane” when used todescribe the location of a polypeptide means that the polypeptideincludes at least one transmembrane domain that traverses the plasmamembrane and at least one domain (e.g., at least one antigen-bindingdomain) that is located in the extracellular space.

The term “hormone receptor ligand binding domain” means a domain in ahormone receptor polypeptide that binds specifically to a hormone or ahormone analogue. Non-limiting examples of a hormone receptor ligandbinding domains (LBD) are described herein. In some cases, a LBD of anuclear hormone receptor is selected from an estrogen receptor, anecdysone receptor, a PPARγ receptor, a glucocorticoid receptor, anandrogen receptor, a thyroid hormone receptor, a mineralocorticoidreceptor, a progesterone receptor, a vitamin D receptor, a PPARreceptorα, a PPARβ/δ receptor, a pregnane X receptor, a liver Xreceptor, a farnesoid X receptor, a retinoid X receptor, a RAR-relatedorphan receptor, and a retinoic acid receptor. In some cases, apolypeptide chain of a heterodimeric polypeptide of the presentdisclosure comprises a single LBD of a nuclear hormone receptor. In somecases, a polypeptide chain of a heterodimeric polypeptide of the presentdisclosure comprises multiple (e.g., two or more) LBDs of a nuclearhormone receptor. In some cases, a polypeptide chain of a heterodimericpolypeptide of the present disclosure comprises two LBDs of a nuclearhormone receptor. In some cases, a polypeptide chain of a heterodimericpolypeptide of the present disclosure comprises three LBDs of a nuclearhormone receptor. Where a polypeptide chain of a heterodimericpolypeptide of the present disclosure comprises multiple (e.g., two ormore) LBDs of a nuclear hormone receptor, in some cases the multipleLBDs comprise identical amino acid sequences. In some cases, the two ormore LBD are in tandem, either directly or separated by a linker (e.g.,any of the linkers described herein).

The term “antigen-binding domain” means a domain that binds specificallyto a target antigen. In some examples, an antigen-binding domain can beformed from the amino acids present within a single-chain polypeptide.In other examples, an antigen-binding domain can be formed from aminoacids present within a first single-chain polypeptide and the aminoacids present in one or more additional single-chain polypeptides (e.g.,a second single-chain polypeptide). Non-limiting examples ofantigen-binding domains are described herein, including, withoutlimitation, scFvs, or LBDs of growth factors. Additional examples ofantigen-binding domains are known in the art.

As used herein, the term “antigen” refers generally to a binding partnerspecifically recognized by an antigen-binding domain described herein.Exemplary antigens include different classes of molecules, such as, butnot limited to, polypeptides and peptide fragments thereof, smallmolecules, lipids, carbohydrates, and nucleic acids. Non-limitingexamples of antigen or antigens that can be specifically bound by any ofthe antigen-binding domains are described herein. Additional examples ofantigen or antigens that can be specifically bound by any of theantigen-binding domains are known in the art.

The term “costimulatory domain” means a signaling domain from anendogenous co-stimulatory transmembrane polypeptide expressed in a Tlymphocyte that promotes the downstream T-cell receptor signaling and/orT cell activation. Non-limiting examples of costimulatory domains aredescribed herein. Additional examples of costimulatory domains are knownin the art. See, e.g., Chen et al., Nature Reviews Immunol. 13:227-242,2013.

The term “immunoreceptor tyrosine-based activation motif” or “ITAM)”means an amino acid motif that includes a four amino-acid consensussequence of a tyrosine separated from a leucine or an isoleucine by twoother amino acids (YxxL/I). The tyrosine residue in the four-amino acidconsensus sequence becomes phosphorylated following interaction of asignaling pathway kinase (e.g., a lymphocyte signaling pathway kinase).Non-limiting examples of ITAMs are described herein. Additional examplesof ITAMs are known in the art.

The term “hormone” means a naturally-occurring molecule thatspecifically binds to a ligand binding domain of a hormone receptor andregulates the activity and/or location of the hormone receptor.Non-limiting examples of hormones are described herein. Additionalexamples of hormones are known in the art.

The term “hormone analogue” means a molecule that mimics the structureof a hormone and specifically binds to a ligand binding domain of ahormone receptor. Non-limiting examples of hormone analogues aredescribed herein. Additional examples of hormone analogues are known inthe art.

The phrase “treatment of cancer” means a reduction in the number,frequency, or severity of one or more (e.g., two, three, four, or five)symptoms of a cancer in a subject having a cancer. Non-limiting symptomsof cancer are described herein. Additional symptoms of cancer are knownin the art.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram of different constructs tested. The labelsextracellular and intracellular refer to the cell membrane, which isdepicted as a gray box between these labels. The extracellular scFvdomain, and intracellular costimulatory (“Costim”), CD3zeta, and mCherrydomains are indicated.

FIG. 2A and FIG. 2B are a set of flow cytometry dot plots for cellsexpressing one of the different constructs. The cells were either leftuntreated or were treated with 500 ng/mL 4-hydroxytamoxifen (“4-OHT”).FIG. 2A is a set of flow cytometry dot plots of Jurkat T cellstransduced with lentiviral vectors encoding the canonical CAR or withanti-CD19 (FMC63) ERa-LBD containing the v2, v3, and v4 CAR constructs.FIG. 2B is a set of flow cytometry dot plots of primary CD4 and CD8human T cells were transduced with lentiviral vectors encoding the v4CAR construct.

FIG. 3 is a set of flow cytometry dot plots for HEK-293T cellstransduced with lentiviral vectors encoding ERa-LBD-containing FMC63 CARfusions. The cells were either left untreated or were treated with 500ng/mL 4-OHT.

FIG. 4 is a set of flow cytometry dot plots for primary human CD4 Tcells transduced with lentiviral vectors encoding a FMC63 NHR LBD-CARfusions and a NHR LBD-CAR having the J6M0 scFv. The cells were eitherleft untreated or were treated with 500 ng/mL 4-OHT.

FIG. 5A is a diagram of the V5 nuclear hormone receptor ligand bindingdomain NHR-LBD CAR construct lacking the costimulatory and CD3zetadomains, and a set of flow cytometry dot plots for cells transduced witha lentiviral vector encoding the V5 construct. The cells were eitherleft untreated or were treated with 500 ng/mL 4-OHT.

FIG. 5B is a set of flow cytometry diagrams of untransduced Jurkat Tcells or Jurkat T cells transduced with a lentiviral vector encoding aCAR in which the scFv domain was replaced with a FLAG tag. Thetransduced cells were either left untreated or were treated with 500ng/mL 4-OHT.

FIG. 6 is a set of flow cytometry diagrams of Jurkat T cells transducedwith CAR constructs having progesterone receptor LBDs. The cells wereeither left untreated or were treated with 500 ng/mL mifepristone.

FIG. 7A is a graph showing T cell activation of primary human T cellstransduced with lentiviral vectors encoding J6M0-ERa-LBD CAR constructafter overnight co-culture with antigen-negative (K562 cell line) andantigen-positive (MM1S cell line) targets.

FIG. 7B is a graph showing IFN-γ production by primary human T cellstransduced with lentiviral vectors encoding J6M0-ERa-LBD CAR constructafter overnight co-culture with antigen-negative (K562 cell line) andantigen-positive (MM1S cell line) targets.

FIG. 8A is a set of flow cytometry diagrams showing proliferation ofprimary human CD4 T cells that were transduced with lentiviral vectorsencoding V4 FMC63-ERa-LBD CAR or canonical CAR controls, co-culturedwith either antigen-negative (K562 cell line) or antigen-positive (Rajicell line) targets, and with increasing concentrations of ligand 4-OHT.

FIG. 8B is graph showing lysis of antigen-positive targets (MM1S cellline) mediated by primary human T cells transduced with lentiviralvectors encoding J6M0-ERa-LBD CAR constructs with increasingconcentrations of 4-OHT.

FIG. 9 is a graph showing surface expression of various CAR constructshaving a LBD at different locations relative to the cell membrane atincreasing concentrations of 4-OHT.

FIG. 10A is a schematic showing timeline of the experimental procedure.

FIG. 10B is a bar graph showing surface expression of various CARconstructs before and after co-culture with 4-OHT.

FIG. 11A is a set of flow cytometry diagrams showing Jurkat T cells thatwere transduced with the FLAG-ERa-LBD CAR, and left untreated or treatedwith 4-OHT for 15 or 120 minutes.

FIG. 11B is a set of flow cytometry diagrams showing human T cells thatwere transduced with lentiviral vector encoding the FMC63-ERa-LBD CAR,and left untreated or treated with 4-OHT for 2 hours, 4 hours, or 24hours.

FIG. 12 is a set of flow cytometry diagrams of primary human T cellsexpressing FMC63-CARs having ERa LBDs. The cells were either leftuntreated or were treated with 500 ng/mL 4-OHT. Some samples werepretreated with 1 μM Brefeldin A.

FIG. 13 is a diagram of the v2 CAR construct and ECD.v1 CAR (also calledLBD-ECD CAR) construct tested. The cellular membrane is depicted as agray box. The extracellular scFv domain, the extracellular ERa LBD, thetransmembrane domain (embedded in the cellular membrane), theintracellular 4-1BB domain, and the intracellular CD3γ domain aredepicted.

FIG. 14 depicts a set of flow cytometry dot plots for cells expressingthe v2 CAR construct or LBD-ECD CAR construct. The cells were eitherleft untreated or were treated with 500 ng/mL 4-hydroxytamoxifen(“4-OHT”).

FIG. 15 is a graph showing the destruction of Nalm 6 tumor cells (%lysis) by T cells transduced with a lentiviral vector encoding LBD-ECDCAR construct (ECD.v1). Cells were treated with between 1.5 ng/mL to 500ng/mL 4-OHT. The positive control was T cells transduced with alentiviral vector encoding constitutive CAR. The negative control was Tcells with LBD-ECD CAR that were not treated with 4-OHT.

FIG. 16 depicts a set of flow cytometry dot plots for cells expressing aconstitutive CAR, LBD-ECD CAR, or v2 CAR. Cells were either leftuntreated, or stimulated with beads containing a CD3/CD28 for 48 hoursprior to flow cytometry. Beads, Dynabeads aCD3/aCD28, were fromThermoFisher. See, Trickett et al., J. Immunol. Methods 275:251-255,2003. Each construct was fused to a cMyc tag.

FIG. 17 is a graph showing IL-2 production from ECD.v1 CAR-T cells orconventional CAR-Tcells after overnight co-culture of the CAR-bearing Tcells with NALM-6 targets in the presence (500 ng/mL) or absence of4-hydroxytamoxifen.

DETAILED DESCRIPTION

Provided herein are single-chain chimeric polypeptides, single-chainchimeric antigen receptors (CARs), and multi-chain chimeric antigenreceptors; nucleic acids encoding the same; vectors including any ofthese nucleic acids; mammalian cells including any of these vectors; andmethods of reversibly inducing the extracellular membrane localizationand methods of altering the membrane localization of these single-chainchimeric polypeptides, single-chain chimeric antigen receptors, andmulti-chain chimeric polypeptides.

Also provided herein are genetically modified mammalian cells having achimeric antigen receptor (CAR). In some cases, a genetically modifiedmammalian cell is derived from a stem cell, a progenitor cell, or a cellderived from a stem cell or a progenitor cell. In some cases, themammalian cell is a T lymphocyte or an NK cell.

Non-limiting aspects of these single-chain chimeric polypeptides,single-chain chimeric antigen receptors, and multi-chain chimericantigen receptors, nucleic acids, vectors, mammalian cells, and methodsare described below, and can be used in any combination withoutlimitation. Additional aspects of these single-chain chimericpolypeptides, single-chain chimeric antigen receptors, and multi-chainchimeric antigen receptors, nucleic acids, vectors, mammalian cells, andmethods are known in the art.

In some embodiments, compositions and/or methods disclosed herein can beused to selectively kill cancer cells in a subject in the presence of ahormone or hormone analogue which binds to an LBD present in thepolypeptide (e.g., a single-chain chimeric polypeptide, a single-chainchimeric antigen receptor, and/or a multi-chain chimeric antigenreceptor). In some embodiments, in the presence of the hormone orhormone analogue, the polypeptide (e.g., the single-chain chimericpolypeptide, the single-chain chimeric antigen receptor, and/or themulti-chain chimeric antigen receptor) localizes to the extracellularsurface, presenting an antigen binding domain (e.g., an scFv or otherantigen-binding domain described herein or known in the art) on theexterior of the cell. The polypeptide (e.g., single-chain chimericpolypeptide, single-chain chimeric antigen receptor, or multi-chainchimeric antigen receptor) can recognize the antigen to which it binds,and thus can selectively kill a target cell expressing the antigen onits surface (e.g., a cancer cell).

In some embodiments, localization of the polypeptide (e.g., thesingle-chain chimeric polypeptide, the single-chain chimeric antigenreceptor, or the multi-chain chimeric antigen receptor) to theextracellular surface can cause toxicity in the subject. In someembodiments, a subject experiencing toxicity to due hormone orhormone-analogue-induced localization of the polypeptide (e.g., thesingle-chain chimeric polypeptide, the single-chain chimeric antigenreceptor, or the multi-chain chimeric antigen receptor) to theextracellular surface can be treated or ameliorated by reducing the doseof the hormone or hormone analogue, or eliminating hormone or hormoneanalogue treatment of the subject altogether. Such reduction in dose orelimination of hormone or hormone analogue treatment can cause thepolypeptide (e.g., the single-chain chimeric polypeptide, thesingle-chain chimeric antigen receptor, or the multi-chain chimericpolypeptide) to be removed from the cell surface and reduce or eliminatepresentation of the antigen binding domain on the cell surface, thusreducing or eliminating toxicity in the subject.

Single-Chain Chimeric Polypeptide

Single-Chain Chimeric Polypeptide—Intracellular Hormone Receptor LBD

Provided herein are single-chain chimeric polypeptides that include atransmembrane domain (e.g., any of the transmembrane domains describedherein or known in the art) and a hormone receptor ligand binding domain(e.g., any of the hormone receptor ligand binding domains describedherein or known in the art), where the transmembrane domain and thehormone receptor ligand binding domain directly abut each other or areseparated by 1 to about 800 amino acids (e.g., or any of the subrangesof this range described herein), and the transmembrane domain and thehormone receptor ligand binding domain are not both present in the same(a single) endogenous single-chain polypeptide in a mammal (e.g., ahuman).

In some examples of the single-chain chimeric polypeptides, thetransmembrane domain directly abuts the hormone receptor ligand bindingdomain (i.e., no intervening amino acids between the transmembranedomain and the hormone receptor ligand binding domain). In some examplesof the single-chain chimeric polypeptides, the transmembrane domain andthe hormone receptor ligand binding domain are separated by: 1 aminoacid to about 800 amino acids, about 780 amino acids, about 760 aminoacids, about 740 amino acids, about 720 amino acids, about 700 aminoacids, about 680 amino acids, about 660 amino acids, about 640 aminoacids, about 620 amino acids, about 600 amino acids, about 580 aminoacids, about 560 amino acids, about 540 amino acids, about 520 aminoacids, about 500 amino acids, about 490 amino acids, about 480 aminoacids, about 470 amino acids, about 460 amino acids, about 450 aminoacids, about 440 amino acids, about 430 amino acids, about 420 aminoacids, about 410 amino acids, about 400 amino acids, about 390 aminoacids, about 380 amino acids, about 370 amino acids, about 360 aminoacids, about 350 amino acids, about 340 amino acids, about 330 aminoacids, about 320 amino acids, about 310 amino acids, about 300 aminoacids, about 290 amino acids, about 280 amino acids, about 270 aminoacids, about 260 amino acids, about 250 amino acids, about 240 aminoacids, about 230 amino acids, about 220 amino acids, about 210 aminoacids, about 200 amino acids, about 195 amino acids, about 190 aminoacids, about 185 amino acids, about 180 amino acids, about 175 aminoacids, about 170 amino acids, about 165 amino acids, about 160 aminoacids, about 155 amino acids, about 150 amino acids, about 145 aminoacids, about 140 amino acids, about 135 amino acids, about 130 aminoacids, about 125 amino acids, about 120 amino acids, about 115 aminoacids, about 110 amino acids, about 105 amino acids, about 100 aminoacids, about 95 amino acids, about 90 amino acids, about 85 amino acids,about 80 amino acids, about 75 amino acids, about 70 amino acids, about65 amino acids, about 60 amino acids, about 55 amino acids, about 50amino acids, about 45 amino acids, about 40 amino acids, about 35 aminoacids, about 30 amino acids, about 25 amino acids, about 20 amino acids,about 18 amino acids, about 16 amino acids, about 14 amino acids, about12 amino acids, about 10 amino acids, about 8 amino acids, about 6 aminoacids, about 5 amino acids, about 4 amino acids, or about 3 amino acids(inclusive); about 2 amino acids to about 800 amino acids, about 780amino acids, about 760 amino acids, about 740 amino acids, about 720amino acids, about 700 amino acids, about 680 amino acids, about 660amino acids, about 640 amino acids, about 620 amino acids, about 600amino acids, about 580 amino acids, about 560 amino acids, about 540amino acids, about 520 amino acids, about 500 amino acids, about 490amino acids, about 480 amino acids, about 470 amino acids, about 460amino acids, about 450 amino acids, about 440 amino acids, about 430amino acids, about 420 amino acids, about 410 amino acids, about 400amino acids, about 390 amino acids, about 380 amino acids, about 370amino acids, about 360 amino acids, about 350 amino acids, about 340amino acids, about 330 amino acids, about 320 amino acids, about 310amino acids, about 300 amino acids, about 290 amino acids, about 280amino acids, about 270 amino acids, about 260 amino acids, about 250amino acids, about 240 amino acids, about 230 amino acids, about 220amino acids, about 210 amino acids, about 200 amino acids, about 195amino acids, about 190 amino acids, about 185 amino acids, about 180amino acids, about 175 amino acids, about 170 amino acids, about 165amino acids, about 160 amino acids, about 155 amino acids, about 150amino acids, about 145 amino acids, about 140 amino acids, about 135amino acids, about 130 amino acids, about 125 amino acids, about 120amino acids, about 115 amino acids, about 110 amino acids, about 105amino acids, about 100 amino acids, about 95 amino acids, about 90 aminoacids, about 85 amino acids, about 80 amino acids, about 75 amino acids,about 70 amino acids, about 65 amino acids, about 60 amino acids, about55 amino acids, about 50 amino acids, about 45 amino acids, about 40amino acids, about 35 amino acids, about 30 amino acids, about 25 aminoacids, about 20 amino acids, about 18 amino acids, about 16 amino acids,about 14 amino acids, about 12 amino acids, about 10 amino acids, about8 amino acids, about 6 amino acids, about 5 amino acids, or about 4amino acids (inclusive); about 3 amino acids to about 800 amino acids,about 780 amino acids, about 760 amino acids, about 740 amino acids,about 720 amino acids, about 700 amino acids, about 680 amino acids,about 660 amino acids, about 640 amino acids, about 620 amino acids,about 600 amino acids, about 580 amino acids, about 560 amino acids,about 540 amino acids, about 520 amino acids, about 500 amino acids,about 490 amino acids, about 480 amino acids, about 470 amino acids,about 460 amino acids, about 450 amino acids, about 440 amino acids,about 430 amino acids, about 420 amino acids, about 410 amino acids,about 400 amino acids, about 390 amino acids, about 380 amino acids,about 370 amino acids, about 360 amino acids, about 350 amino acids,about 340 amino acids, about 330 amino acids, about 320 amino acids,about 310 amino acids, about 300 amino acids, about 290 amino acids,about 280 amino acids, about 270 amino acids, about 260 amino acids,about 250 amino acids, about 240 amino acids, about 230 amino acids,about 220 amino acids, about 210 amino acids, about 200 amino acids,about 195 amino acids, about 190 amino acids, about 185 amino acids,about 180 amino acids, about 175 amino acids, about 170 amino acids,about 165 amino acids, about 160 amino acids, about 155 amino acids,about 150 amino acids, about 145 amino acids, about 140 amino acids,about 135 amino acids, about 130 amino acids, about 125 amino acids,about 120 amino acids, about 115 amino acids, about 110 amino acids,about 105 amino acids, about 100 amino acids, about 95 amino acids,about 90 amino acids, about 85 amino acids, about 80 amino acids, about75 amino acids, about 70 amino acids, about 65 amino acids, about 60amino acids, about 55 amino acids, about 50 amino acids, about 45 aminoacids, about 40 amino acids, about 35 amino acids, about 30 amino acids,about 25 amino acids, about 20 amino acids, about 18 amino acids, about16 amino acids, about 14 amino acids, about 12 amino acids, about 10amino acids, about 8 amino acids, about 6 amino acids, or about 5 aminoacids (inclusive); about 4 amino acids to about 800 amino acids, about780 amino acids, about 760 amino acids, about 740 amino acids, about 720amino acids, about 700 amino acids, about 680 amino acids, about 660amino acids, about 640 amino acids, about 620 amino acids, about 600amino acids, about 580 amino acids, about 560 amino acids, about 540amino acids, about 520 amino acids, about 500 amino acids, about 490amino acids, about 480 amino acids, about 470 amino acids, about 460amino acids, about 450 amino acids, about 440 amino acids, about 430amino acids, about 420 amino acids, about 410 amino acids, about 400amino acids, about 390 amino acids, about 380 amino acids, about 370amino acids, about 360 amino acids, about 350 amino acids, about 340amino acids, about 330 amino acids, about 320 amino acids, about 310amino acids, about 300 amino acids, about 290 amino acids, about 280amino acids, about 270 amino acids, about 260 amino acids, about 250amino acids, about 240 amino acids, about 230 amino acids, about 220amino acids, about 210 amino acids, about 200 amino acids, about 195amino acids, about 190 amino acids, about 185 amino acids, about 180amino acids, about 175 amino acids, about 170 amino acids, about 165amino acids, about 160 amino acids, about 155 amino acids, about 150amino acids, about 145 amino acids, about 140 amino acids, about 135amino acids, about 130 amino acids, about 125 amino acids, about 120amino acids, about 115 amino acids, about 110 amino acids, about 105amino acids, about 100 amino acids, about 95 amino acids, about 90 aminoacids, about 85 amino acids, about 80 amino acids, about 75 amino acids,about 70 amino acids, about 65 amino acids, about 60 amino acids, about55 amino acids, about 50 amino acids, about 45 amino acids, about 40amino acids, about 35 amino acids, about 30 amino acids, about 25 aminoacids, about 20 amino acids, about 18 amino acids, about 16 amino acids,about 14 amino acids, about 12 amino acids, about 10 amino acids, about8 amino acids, or about 6 amino acids (inclusive); about 5 amino acidsto about 800 amino acids, about 780 amino acids, about 760 amino acids,about 740 amino acids, about 720 amino acids, about 700 amino acids,about 680 amino acids, about 660 amino acids, about 640 amino acids,about 620 amino acids, about 600 amino acids, about 580 amino acids,about 560 amino acids, about 540 amino acids, about 520 amino acids,about 500 amino acids, about 490 amino acids, about 480 amino acids,about 470 amino acids, about 460 amino acids, about 450 amino acids,about 440 amino acids, about 430 amino acids, about 420 amino acids,about 410 amino acids, about 400 amino acids, about 390 amino acids,about 380 amino acids, about 370 amino acids, about 360 amino acids,about 350 amino acids, about 340 amino acids, about 330 amino acids,about 320 amino acids, about 310 amino acids, about 300 amino acids,about 290 amino acids, about 280 amino acids, about 270 amino acids,about 260 amino acids, about 250 amino acids, about 240 amino acids,about 230 amino acids, about 220 amino acids, about 210 amino acids,about 200 amino acids, about 195 amino acids, about 190 amino acids,about 185 amino acids, about 180 amino acids, about 175 amino acids,about 170 amino acids, about 165 amino acids, about 160 amino acids,about 155 amino acids, about 150 amino acids, about 145 amino acids,about 140 amino acids, about 135 amino acids, about 130 amino acids,about 125 amino acids, about 120 amino acids, about 115 amino acids,about 110 amino acids, about 105 amino acids, about 100 amino acids,about 95 amino acids, about 90 amino acids, about 85 amino acids, about80 amino acids, about 75 amino acids, about 70 amino acids, about 65amino acids, about 60 amino acids, about 55 amino acids, about 50 aminoacids, about 45 amino acids, about 40 amino acids, about 35 amino acids,about 30 amino acids, about 25 amino acids, about 20 amino acids, about18 amino acids, about 16 amino acids, about 14 amino acids, about 12amino acids, about 10 amino acids, or about 8 amino acids (inclusive);about 6 amino acids to about 800 amino acids, about 780 amino acids,about 760 amino acids, about 740 amino acids, about 720 amino acids,about 700 amino acids, about 680 amino acids, about 660 amino acids,about 640 amino acids, about 620 amino acids, about 600 amino acids,about 580 amino acids, about 560 amino acids, about 540 amino acids,about 520 amino acids, about 500 amino acids, about 490 amino acids,about 480 amino acids, about 470 amino acids, about 460 amino acids,about 450 amino acids, about 440 amino acids, about 430 amino acids,about 420 amino acids, about 410 amino acids, about 400 amino acids,about 390 amino acids, about 380 amino acids, about 370 amino acids,about 360 amino acids, about 350 amino acids, about 340 amino acids,about 330 amino acids, about 320 amino acids, about 310 amino acids,about 300 amino acids, about 290 amino acids, about 280 amino acids,about 270 amino acids, about 260 amino acids, about 250 amino acids,about 240 amino acids, about 230 amino acids, about 220 amino acids,about 210 amino acids, about 200 amino acids, about 195 amino acids,about 190 amino acids, about 185 amino acids, about 180 amino acids,about 175 amino acids, about 170 amino acids, about 165 amino acids,about 160 amino acids, about 155 amino acids, about 150 amino acids,about 145 amino acids, about 140 amino acids, about 135 amino acids,about 130 amino acids, about 125 amino acids, about 120 amino acids,about 115 amino acids, about 110 amino acids, about 105 amino acids,about 100 amino acids, about 95 amino acids, about 90 amino acids, about85 amino acids, about 80 amino acids, about 75 amino acids, about 70amino acids, about 65 amino acids, about 60 amino acids, about 55 aminoacids, about 50 amino acids, about 45 amino acids, about 40 amino acids,about 35 amino acids, about 30 amino acids, about 25 amino acids, about20 amino acids, about 18 amino acids, about 16 amino acids, about 14amino acids, about 12 amino acids, about 10 amino acids, or about 8amino acids (inclusive); about 8 amino acids to about 800 amino acids,about 780 amino acids, about 760 amino acids, about 740 amino acids,about 720 amino acids, about 700 amino acids, about 680 amino acids,about 660 amino acids, about 640 amino acids, about 620 amino acids,about 600 amino acids, about 580 amino acids, about 560 amino acids,about 540 amino acids, about 520 amino acids, about 500 amino acids,about 490 amino acids, about 480 amino acids, about 470 amino acids,about 460 amino acids, about 450 amino acids, about 440 amino acids,about 430 amino acids, about 420 amino acids, about 410 amino acids,about 400 amino acids, about 390 amino acids, about 380 amino acids,about 370 amino acids, about 360 amino acids, about 350 amino acids,about 340 amino acids, about 330 amino acids, about 320 amino acids,about 310 amino acids, about 300 amino acids, about 290 amino acids,about 280 amino acids, about 270 amino acids, about 260 amino acids,about 250 amino acids, about 240 amino acids, about 230 amino acids,about 220 amino acids, about 210 amino acids, about 200 amino acids,about 195 amino acids, about 190 amino acids, about 185 amino acids,about 180 amino acids, about 175 amino acids, about 170 amino acids,about 165 amino acids, about 160 amino acids, about 155 amino acids,about 150 amino acids, about 145 amino acids, about 140 amino acids,about 135 amino acids, about 130 amino acids, about 125 amino acids,about 120 amino acids, about 115 amino acids, about 110 amino acids,about 105 amino acids, about 100 amino acids, about 95 amino acids,about 90 amino acids, about 85 amino acids, about 80 amino acids, about75 amino acids, about 70 amino acids, about 65 amino acids, about 60amino acids, about 55 amino acids, about 50 amino acids, about 45 aminoacids, about 40 amino acids, about 35 amino acids, about 30 amino acids,about 25 amino acids, about 20 amino acids, about 18 amino acids, about16 amino acids, about 14 amino acids, about 12 amino acids, or about 10amino acids (inclusive); about 10 amino acids to about 800 amino acids,about 780 amino acids, about 760 amino acids, about 740 amino acids,about 720 amino acids, about 700 amino acids, about 680 amino acids,about 660 amino acids, about 640 amino acids, about 620 amino acids,about 600 amino acids, about 580 amino acids, about 560 amino acids,about 540 amino acids, about 520 amino acids, about 500 amino acids,about 490 amino acids, about 480 amino acids, about 470 amino acids,about 460 amino acids, about 450 amino acids, about 440 amino acids,about 430 amino acids, about 420 amino acids, about 410 amino acids,about 400 amino acids, about 390 amino acids, about 380 amino acids,about 370 amino acids, about 360 amino acids, about 350 amino acids,about 340 amino acids, about 330 amino acids, about 320 amino acids,about 310 amino acids, about 300 amino acids, about 290 amino acids,about 280 amino acids, about 270 amino acids, about 260 amino acids,about 250 amino acids, about 240 amino acids, about 230 amino acids,about 220 amino acids, about 210 amino acids, about 200 amino acids,about 195 amino acids, about 190 amino acids, about 185 amino acids,about 180 amino acids, about 175 amino acids, about 170 amino acids,about 165 amino acids, about 160 amino acids, about 155 amino acids,about 150 amino acids, about 145 amino acids, about 140 amino acids,about 135 amino acids, about 130 amino acids, about 125 amino acids,about 120 amino acids, about 115 amino acids, about 110 amino acids,about 105 amino acids, about 100 amino acids, about 95 amino acids,about 90 amino acids, about 85 amino acids, about 80 amino acids, about75 amino acids, about 70 amino acids, about 65 amino acids, about 60amino acids, about 55 amino acids, about 50 amino acids, about 45 aminoacids, about 40 amino acids, about 35 amino acids, about 30 amino acids,about 25 amino acids, about 20 amino acids, about 18 amino acids, about16 amino acids, about 14 amino acids, or about 12 amino acids(inclusive); about 12 amino acids to about 800 amino acids, about 780amino acids, about 760 amino acids, about 740 amino acids, about 720amino acids, about 700 amino acids, about 680 amino acids, about 660amino acids, about 640 amino acids, about 620 amino acids, about 600amino acids, about 580 amino acids, about 560 amino acids, about 540amino acids, about 520 amino acids, about 500 amino acids, about 490amino acids, about 480 amino acids, about 470 amino acids, about 460amino acids, about 450 amino acids, about 440 amino acids, about 430amino acids, about 420 amino acids, about 410 amino acids, about 400amino acids, about 390 amino acids, about 380 amino acids, about 370amino acids, about 360 amino acids, about 350 amino acids, about 340amino acids, about 330 amino acids, about 320 amino acids, about 310amino acids, about 300 amino acids, about 290 amino acids, about 280amino acids, about 270 amino acids, about 260 amino acids, about 250amino acids, about 240 amino acids, about 230 amino acids, about 220amino acids, about 210 amino acids, about 200 amino acids, about 195amino acids, about 190 amino acids, about 185 amino acids, about 180amino acids, about 175 amino acids, about 170 amino acids, about 165amino acids, about 160 amino acids, about 155 amino acids, about 150amino acids, about 145 amino acids, about 140 amino acids, about 135amino acids, about 130 amino acids, about 125 amino acids, about 120amino acids, about 115 amino acids, about 110 amino acids, about 105amino acids, about 100 amino acids, about 95 amino acids, about 90 aminoacids, about 85 amino acids, about 80 amino acids, about 75 amino acids,about 70 amino acids, about 65 amino acids, about 60 amino acids, about55 amino acids, about 50 amino acids, about 45 amino acids, about 40amino acids, about 35 amino acids, about 30 amino acids, about 25 aminoacids, about 20 amino acids, about 18 amino acids, about 16 amino acids,or about 14 amino acids (inclusive); about 14 amino acids to about 800amino acids, about 780 amino acids, about 760 amino acids, about 740amino acids, about 720 amino acids, about 700 amino acids, about 680amino acids, about 660 amino acids, about 640 amino acids, about 620amino acids, about 600 amino acids, about 580 amino acids, about 560amino acids, about 540 amino acids, about 520 amino acids, about 500amino acids, about 490 amino acids, about 480 amino acids, about 470amino acids, about 460 amino acids, about 450 amino acids, about 440amino acids, about 430 amino acids, about 420 amino acids, about 410amino acids, about 400 amino acids, about 390 amino acids, about 380amino acids, about 370 amino acids, about 360 amino acids, about 350amino acids, about 340 amino acids, about 330 amino acids, about 320amino acids, about 310 amino acids, about 300 amino acids, about 290amino acids, about 280 amino acids, about 270 amino acids, about 260amino acids, about 250 amino acids, about 240 amino acids, about 230amino acids, about 220 amino acids, about 210 amino acids, about 200amino acids, about 195 amino acids, about 190 amino acids, about 185amino acids, about 180 amino acids, about 175 amino acids, about 170amino acids, about 165 amino acids, about 160 amino acids, about 155amino acids, about 150 amino acids, about 145 amino acids, about 140amino acids, about 135 amino acids, about 130 amino acids, about 125amino acids, about 120 amino acids, about 115 amino acids, about 110amino acids, about 105 amino acids, about 100 amino acids, about 95amino acids, about 90 amino acids, about 85 amino acids, about 80 aminoacids, about 75 amino acids, about 70 amino acids, about 65 amino acids,about 60 amino acids, about 55 amino acids, about 50 amino acids, about45 amino acids, about 40 amino acids, about 35 amino acids, about 30amino acids, about 25 amino acids, about 20 amino acids, about 18 aminoacids, or about 16 amino acids (inclusive); about 16 amino acids toabout 800 amino acids, about 780 amino acids, about 760 amino acids,about 740 amino acids, about 720 amino acids, about 700 amino acids,about 680 amino acids, about 660 amino acids, about 640 amino acids,about 620 amino acids, about 600 amino acids, about 580 amino acids,about 560 amino acids, about 540 amino acids, about 520 amino acids,about 500 amino acids, about 490 amino acids, about 480 amino acids,about 470 amino acids, about 460 amino acids, about 450 amino acids,about 440 amino acids, about 430 amino acids, about 420 amino acids,about 410 amino acids, about 400 amino acids, about 390 amino acids,about 380 amino acids, about 370 amino acids, about 360 amino acids,about 350 amino acids, about 340 amino acids, about 330 amino acids,about 320 amino acids, about 310 amino acids, about 300 amino acids,about 290 amino acids, about 280 amino acids, about 270 amino acids,about 260 amino acids, about 250 amino acids, about 240 amino acids,about 230 amino acids, about 220 amino acids, about 210 amino acids,about 200 amino acids, about 195 amino acids, about 190 amino acids,about 185 amino acids, about 180 amino acids, about 175 amino acids,about 170 amino acids, about 165 amino acids, about 160 amino acids,about 155 amino acids, about 150 amino acids, about 145 amino acids,about 140 amino acids, about 135 amino acids, about 130 amino acids,about 125 amino acids, about 120 amino acids, about 115 amino acids,about 110 amino acids, about 105 amino acids, about 100 amino acids,about 95 amino acids, about 90 amino acids, about 85 amino acids, about80 amino acids, about 75 amino acids, about 70 amino acids, about 65amino acids, about 60 amino acids, about 55 amino acids, about 50 aminoacids, about 45 amino acids, about 40 amino acids, about 35 amino acids,about 30 amino acids, about 25 amino acids, about 20 amino acids, orabout 18 amino acids (inclusive); about 18 amino acids to about 800amino acids, about 780 amino acids, about 760 amino acids, about 740amino acids, about 720 amino acids, about 700 amino acids, about 680amino acids, about 660 amino acids, about 640 amino acids, about 620amino acids, about 600 amino acids, about 580 amino acids, about 560amino acids, about 540 amino acids, about 520 amino acids, about 500amino acids, about 490 amino acids, about 480 amino acids, about 470amino acids, about 460 amino acids, about 450 amino acids, about 440amino acids, about 430 amino acids, about 420 amino acids, about 410amino acids, about 400 amino acids, about 390 amino acids, about 380amino acids, about 370 amino acids, about 360 amino acids, about 350amino acids, about 340 amino acids, about 330 amino acids, about 320amino acids, about 310 amino acids, about 300 amino acids, about 290amino acids, about 280 amino acids, about 270 amino acids, about 260amino acids, about 250 amino acids, about 240 amino acids, about 230amino acids, about 220 amino acids, about 210 amino acids, about 200amino acids, about 195 amino acids, about 190 amino acids, about 185amino acids, about 180 amino acids, about 175 amino acids, about 170amino acids, about 165 amino acids, about 160 amino acids, about 155amino acids, about 150 amino acids, about 145 amino acids, about 140amino acids, about 135 amino acids, about 130 amino acids, about 125amino acids, about 120 amino acids, about 115 amino acids, about 110amino acids, about 105 amino acids, about 100 amino acids, about 95amino acids, about 90 amino acids, about 85 amino acids, about 80 aminoacids, about 75 amino acids, about 70 amino acids, about 65 amino acids,about 60 amino acids, about 55 amino acids, about 50 amino acids, about45 amino acids, about 40 amino acids, about 35 amino acids, about 30amino acids, about 25 amino acids, or about 20 amino acids (inclusive);about 20 amino acids to about 800 amino acids, about 780 amino acids,about 760 amino acids, about 740 amino acids, about 720 amino acids,about 700 amino acids, about 680 amino acids, about 660 amino acids,about 640 amino acids, about 620 amino acids, about 600 amino acids,about 580 amino acids, about 560 amino acids, about 540 amino acids,about 520 amino acids, about 500 amino acids, about 490 amino acids,about 480 amino acids, about 470 amino acids, about 460 amino acids,about 450 amino acids, about 440 amino acids, about 430 amino acids,about 420 amino acids, about 410 amino acids, about 400 amino acids,about 390 amino acids, about 380 amino acids, about 370 amino acids,about 360 amino acids, about 350 amino acids, about 340 amino acids,about 330 amino acids, about 320 amino acids, about 310 amino acids,about 300 amino acids, about 290 amino acids, about 280 amino acids,about 270 amino acids, about 260 amino acids, about 250 amino acids,about 240 amino acids, about 230 amino acids, about 220 amino acids,about 210 amino acids, about 200 amino acids, about 195 amino acids,about 190 amino acids, about 185 amino acids, about 180 amino acids,about 175 amino acids, about 170 amino acids, about 165 amino acids,about 160 amino acids, about 155 amino acids, about 150 amino acids,about 145 amino acids, about 140 amino acids, about 135 amino acids,about 130 amino acids, about 125 amino acids, about 120 amino acids,about 115 amino acids, about 110 amino acids, about 105 amino acids,about 100 amino acids, about 95 amino acids, about 90 amino acids, about85 amino acids, about 80 amino acids, about 75 amino acids, about 70amino acids, about 65 amino acids, about 60 amino acids, about 55 aminoacids, about 50 amino acids, about 45 amino acids, about 40 amino acids,about 35 amino acids, about 30 amino acids, or about 25 amino acids(inclusive); about 25 amino acids to about 800 amino acids, about 780amino acids, about 760 amino acids, about 740 amino acids, about 720amino acids, about 700 amino acids, about 680 amino acids, about 660amino acids, about 640 amino acids, about 620 amino acids, about 600amino acids, about 580 amino acids, about 560 amino acids, about 540amino acids, about 520 amino acids, about 500 amino acids, about 490amino acids, about 480 amino acids, about 470 amino acids, about 460amino acids, about 450 amino acids, about 440 amino acids, about 430amino acids, about 420 amino acids, about 410 amino acids, about 400amino acids, about 390 amino acids, about 380 amino acids, about 370amino acids, about 360 amino acids, about 350 amino acids, about 340amino acids, about 330 amino acids, about 320 amino acids, about 310amino acids, about 300 amino acids, about 290 amino acids, about 280amino acids, about 270 amino acids, about 260 amino acids, about 250amino acids, about 240 amino acids, about 230 amino acids, about 220amino acids, about 210 amino acids, about 200 amino acids, about 195amino acids, about 190 amino acids, about 185 amino acids, about 180amino acids, about 175 amino acids, about 170 amino acids, about 165amino acids, about 160 amino acids, about 155 amino acids, about 150amino acids, about 145 amino acids, about 140 amino acids, about 135amino acids, about 130 amino acids, about 125 amino acids, about 120amino acids, about 115 amino acids, about 110 amino acids, about 105amino acids, about 100 amino acids, about 95 amino acids, about 90 aminoacids, about 85 amino acids, about 80 amino acids, about 75 amino acids,about 70 amino acids, about 65 amino acids, about 60 amino acids, about55 amino acids, about 50 amino acids, about 45 amino acids, about 40amino acids, about 35 amino acids, or about 30 amino acids (inclusive);about 30 amino acids to about 800 amino acids, about 780 amino acids,about 760 amino acids, about 740 amino acids, about 720 amino acids,about 700 amino acids, about 680 amino acids, about 660 amino acids,about 640 amino acids, about 620 amino acids, about 600 amino acids,about 580 amino acids, about 560 amino acids, about 540 amino acids,about 520 amino acids, about 500 amino acids, about 490 amino acids,about 480 amino acids, about 470 amino acids, about 460 amino acids,about 450 amino acids, about 440 amino acids, about 430 amino acids,about 420 amino acids, about 410 amino acids, about 400 amino acids,about 390 amino acids, about 380 amino acids, about 370 amino acids,about 360 amino acids, about 350 amino acids, about 340 amino acids,about 330 amino acids, about 320 amino acids, about 310 amino acids,about 300 amino acids, about 290 amino acids, about 280 amino acids,about 270 amino acids, about 260 amino acids, about 250 amino acids,about 240 amino acids, about 230 amino acids, about 220 amino acids,about 210 amino acids, about 200 amino acids, about 195 amino acids,about 190 amino acids, about 185 amino acids, about 180 amino acids,about 175 amino acids, about 170 amino acids, about 165 amino acids,about 160 amino acids, about 155 amino acids, about 150 amino acids,about 145 amino acids, about 140 amino acids, about 135 amino acids,about 130 amino acids, about 125 amino acids, about 120 amino acids,about 115 amino acids, about 110 amino acids, about 105 amino acids,about 100 amino acids, about 95 amino acids, about 90 amino acids, about85 amino acids, about 80 amino acids, about 75 amino acids, about 70amino acids, about 65 amino acids, about 60 amino acids, about 55 aminoacids, about 50 amino acids, about 45 amino acids, about 40 amino acids,or about 35 amino acids (inclusive); about 35 amino acids to about 800amino acids, about 780 amino acids, about 760 amino acids, about 740amino acids, about 720 amino acids, about 700 amino acids, about 680amino acids, about 660 amino acids, about 640 amino acids, about 620amino acids, about 600 amino acids, about 580 amino acids, about 560amino acids, about 540 amino acids, about 520 amino acids, about 500amino acids, about 490 amino acids, about 480 amino acids, about 470amino acids, about 460 amino acids, about 450 amino acids, about 440amino acids, about 430 amino acids, about 420 amino acids, about 410amino acids, about 400 amino acids, about 390 amino acids, about 380amino acids, about 370 amino acids, about 360 amino acids, about 350amino acids, about 340 amino acids, about 330 amino acids, about 320amino acids, about 310 amino acids, about 300 amino acids, about 290amino acids, about 280 amino acids, about 270 amino acids, about 260amino acids, about 250 amino acids, about 240 amino acids, about 230amino acids, about 220 amino acids, about 210 amino acids, about 200amino acids, about 195 amino acids, about 190 amino acids, about 185amino acids, about 180 amino acids, about 175 amino acids, about 170amino acids, about 165 amino acids, about 160 amino acids, about 155amino acids, about 150 amino acids, about 145 amino acids, about 140amino acids, about 135 amino acids, about 130 amino acids, about 125amino acids, about 120 amino acids, about 115 amino acids, about 110amino acids, about 105 amino acids, about 100 amino acids, about 95amino acids, about 90 amino acids, about 85 amino acids, about 80 aminoacids, about 75 amino acids, about 70 amino acids, about 65 amino acids,about 60 amino acids, about 55 amino acids, about 50 amino acids, about45 amino acids, or about 40 amino acids (inclusive); about 40 aminoacids to about 800 amino acids, about 780 amino acids, about 760 aminoacids, about 740 amino acids, about 720 amino acids, about 700 aminoacids, about 680 amino acids, about 660 amino acids, about 640 aminoacids, about 620 amino acids, about 600 amino acids, about 580 aminoacids, about 560 amino acids, about 540 amino acids, about 520 aminoacids, about 500 amino acids, about 490 amino acids, about 480 aminoacids, about 470 amino acids, about 460 amino acids, about 450 aminoacids, about 440 amino acids, about 430 amino acids, about 420 aminoacids, about 410 amino acids, about 400 amino acids, about 390 aminoacids, about 380 amino acids, about 370 amino acids, about 360 aminoacids, about 350 amino acids, about 340 amino acids, about 330 aminoacids, about 320 amino acids, about 310 amino acids, about 300 aminoacids, about 290 amino acids, about 280 amino acids, about 270 aminoacids, about 260 amino acids, about 250 amino acids, about 240 aminoacids, about 230 amino acids, about 220 amino acids, about 210 aminoacids, about 200 amino acids, about 195 amino acids, about 190 aminoacids, about 185 amino acids, about 180 amino acids, about 175 aminoacids, about 170 amino acids, about 165 amino acids, about 160 aminoacids, about 155 amino acids, about 150 amino acids, about 145 aminoacids, about 140 amino acids, about 135 amino acids, about 130 aminoacids, about 125 amino acids, about 120 amino acids, about 115 aminoacids, about 110 amino acids, about 105 amino acids, about 100 aminoacids, about 95 amino acids, about 90 amino acids, about 85 amino acids,about 80 amino acids, about 75 amino acids, about 70 amino acids, about65 amino acids, about 60 amino acids, about 55 amino acids, about 50amino acids, or about 45 amino acids (inclusive); about 45 amino acidsto about 800 amino acids, about 780 amino acids, about 760 amino acids,about 740 amino acids, about 720 amino acids, about 700 amino acids,about 680 amino acids, about 660 amino acids, about 640 amino acids,about 620 amino acids, about 600 amino acids, about 580 amino acids,about 560 amino acids, about 540 amino acids, about 520 amino acids,about 500 amino acids, about 490 amino acids, about 480 amino acids,about 470 amino acids, about 460 amino acids, about 450 amino acids,about 440 amino acids, about 430 amino acids, about 420 amino acids,about 410 amino acids, about 400 amino acids, about 390 amino acids,about 380 amino acids, about 370 amino acids, about 360 amino acids,about 350 amino acids, about 340 amino acids, about 330 amino acids,about 320 amino acids, about 310 amino acids, about 300 amino acids,about 290 amino acids, about 280 amino acids, about 270 amino acids,about 260 amino acids, about 250 amino acids, about 240 amino acids,about 230 amino acids, about 220 amino acids, about 210 amino acids,about 200 amino acids, about 195 amino acids, about 190 amino acids,about 185 amino acids, about 180 amino acids, about 175 amino acids,about 170 amino acids, about 165 amino acids, about 160 amino acids,about 155 amino acids, about 150 amino acids, about 145 amino acids,about 140 amino acids, about 135 amino acids, about 130 amino acids,about 125 amino acids, about 120 amino acids, about 115 amino acids,about 110 amino acids, about 105 amino acids, about 100 amino acids,about 95 amino acids, about 90 amino acids, about 85 amino acids, about80 amino acids, about 75 amino acids, about 70 amino acids, about 65amino acids, about 60 amino acids, about 55 amino acids, or about 50amino acids (inclusive); about 50 amino acids to about 800 amino acids,about 780 amino acids, about 760 amino acids, about 740 amino acids,about 720 amino acids, about 700 amino acids, about 680 amino acids,about 660 amino acids, about 640 amino acids, about 620 amino acids,about 600 amino acids, about 580 amino acids, about 560 amino acids,about 540 amino acids, about 520 amino acids, about 500 amino acids,about 490 amino acids, about 480 amino acids, about 470 amino acids,about 460 amino acids, about 450 amino acids, about 440 amino acids,about 430 amino acids, about 420 amino acids, about 410 amino acids,about 400 amino acids, about 390 amino acids, about 380 amino acids,about 370 amino acids, about 360 amino acids, about 350 amino acids,about 340 amino acids, about 330 amino acids, about 320 amino acids,about 310 amino acids, about 300 amino acids, about 290 amino acids,about 280 amino acids, about 270 amino acids, about 260 amino acids,about 250 amino acids, about 240 amino acids, about 230 amino acids,about 220 amino acids, about 210 amino acids, about 200 amino acids,about 195 amino acids, about 190 amino acids, about 185 amino acids,about 180 amino acids, about 175 amino acids, about 170 amino acids,about 165 amino acids, about 160 amino acids, about 155 amino acids,about 150 amino acids, about 145 amino acids, about 140 amino acids,about 135 amino acids, about 130 amino acids, about 125 amino acids,about 120 amino acids, about 115 amino acids, about 110 amino acids,about 105 amino acids, about 100 amino acids, about 95 amino acids,about 90 amino acids, about 85 amino acids, about 80 amino acids, about75 amino acids, about 70 amino acids, about 65 amino acids, about 60amino acids, or about 55 amino acids (inclusive); about 55 amino acidsto about 800 amino acids, about 780 amino acids, about 760 amino acids,about 740 amino acids, about 720 amino acids, about 700 amino acids,about 680 amino acids, about 660 amino acids, about 640 amino acids,about 620 amino acids, about 600 amino acids, about 580 amino acids,about 560 amino acids, about 540 amino acids, about 520 amino acids,about 500 amino acids, about 490 amino acids, about 480 amino acids,about 470 amino acids, about 460 amino acids, about 450 amino acids,about 440 amino acids, about 430 amino acids, about 420 amino acids,about 410 amino acids, about 400 amino acids, about 390 amino acids,about 380 amino acids, about 370 amino acids, about 360 amino acids,about 350 amino acids, about 340 amino acids, about 330 amino acids,about 320 amino acids, about 310 amino acids, about 300 amino acids,about 290 amino acids, about 280 amino acids, about 270 amino acids,about 260 amino acids, about 250 amino acids, about 240 amino acids,about 230 amino acids, about 220 amino acids, about 210 amino acids,about 200 amino acids, about 195 amino acids, about 190 amino acids,about 185 amino acids, about 180 amino acids, about 175 amino acids,about 170 amino acids, about 165 amino acids, about 160 amino acids,about 155 amino acids, about 150 amino acids, about 145 amino acids,about 140 amino acids, about 135 amino acids, about 130 amino acids,about 125 amino acids, about 120 amino acids, about 115 amino acids,about 110 amino acids, about 105 amino acids, about 100 amino acids,about 95 amino acids, about 90 amino acids, about 85 amino acids, about80 amino acids, about 75 amino acids, about 70 amino acids, about 65amino acids, or about 60 amino acids (inclusive); about 60 amino acidsto about 800 amino acids, about 780 amino acids, about 760 amino acids,about 740 amino acids, about 720 amino acids, about 700 amino acids,about 680 amino acids, about 660 amino acids, about 640 amino acids,about 620 amino acids, about 600 amino acids, about 580 amino acids,about 560 amino acids, about 540 amino acids, about 520 amino acids,about 500 amino acids, about 490 amino acids, about 480 amino acids,about 470 amino acids, about 460 amino acids, about 450 amino acids,about 440 amino acids, about 430 amino acids, about 420 amino acids,about 410 amino acids, about 400 amino acids, about 390 amino acids,about 380 amino acids, about 370 amino acids, about 360 amino acids,about 350 amino acids, about 340 amino acids, about 330 amino acids,about 320 amino acids, about 310 amino acids, about 300 amino acids,about 290 amino acids, about 280 amino acids, about 270 amino acids,about 260 amino acids, about 250 amino acids, about 240 amino acids,about 230 amino acids, about 220 amino acids, about 210 amino acids,about 200 amino acids, about 195 amino acids, about 190 amino acids,about 185 amino acids, about 180 amino acids, about 175 amino acids,about 170 amino acids, about 165 amino acids, about 160 amino acids,about 155 amino acids, about 150 amino acids, about 145 amino acids,about 140 amino acids, about 135 amino acids, about 130 amino acids,about 125 amino acids, about 120 amino acids, about 115 amino acids,about 110 amino acids, about 105 amino acids, about 100 amino acids,about 95 amino acids, about 90 amino acids, about 85 amino acids, about80 amino acids, about 75 amino acids, about 70 amino acids, or about 65amino acids (inclusive); about 65 amino acids to about 800 amino acids,about 780 amino acids, about 760 amino acids, about 740 amino acids,about 720 amino acids, about 700 amino acids, about 680 amino acids,about 660 amino acids, about 640 amino acids, about 620 amino acids,about 600 amino acids, about 580 amino acids, about 560 amino acids,about 540 amino acids, about 520 amino acids, about 500 amino acids,about 490 amino acids, about 480 amino acids, about 470 amino acids,about 460 amino acids, about 450 amino acids, about 440 amino acids,about 430 amino acids, about 420 amino acids, about 410 amino acids,about 400 amino acids, about 390 amino acids, about 380 amino acids,about 370 amino acids, about 360 amino acids, about 350 amino acids,about 340 amino acids, about 330 amino acids, about 320 amino acids,about 310 amino acids, about 300 amino acids, about 290 amino acids,about 280 amino acids, about 270 amino acids, about 260 amino acids,about 250 amino acids, about 240 amino acids, about 230 amino acids,about 220 amino acids, about 210 amino acids, about 200 amino acids,about 195 amino acids, about 190 amino acids, about 185 amino acids,about 180 amino acids, about 175 amino acids, about 170 amino acids,about 165 amino acids, about 160 amino acids, about 155 amino acids,about 150 amino acids, about 145 amino acids, about 140 amino acids,about 135 amino acids, about 130 amino acids, about 125 amino acids,about 120 amino acids, about 115 amino acids, about 110 amino acids,about 105 amino acids, about 100 amino acids, about 95 amino acids,about 90 amino acids, about 85 amino acids, about 80 amino acids, about75 amino acids, or about 70 amino acids (inclusive); about 70 aminoacids to about 800 amino acids, about 780 amino acids, about 760 aminoacids, about 740 amino acids, about 720 amino acids, about 700 aminoacids, about 680 amino acids, about 660 amino acids, about 640 aminoacids, about 620 amino acids, about 600 amino acids, about 580 aminoacids, about 560 amino acids, about 540 amino acids, about 520 aminoacids, about 500 amino acids, about 490 amino acids, about 480 aminoacids, about 470 amino acids, about 460 amino acids, about 450 aminoacids, about 440 amino acids, about 430 amino acids, about 420 aminoacids, about 410 amino acids, about 400 amino acids, about 390 aminoacids, about 380 amino acids, about 370 amino acids, about 360 aminoacids, about 350 amino acids, about 340 amino acids, about 330 aminoacids, about 320 amino acids, about 310 amino acids, about 300 aminoacids, about 290 amino acids, about 280 amino acids, about 270 aminoacids, about 260 amino acids, about 250 amino acids, about 240 aminoacids, about 230 amino acids, about 220 amino acids, about 210 aminoacids, about 200 amino acids, about 195 amino acids, about 190 aminoacids, about 185 amino acids, about 180 amino acids, about 175 aminoacids, about 170 amino acids, about 165 amino acids, about 160 aminoacids, about 155 amino acids, about 150 amino acids, about 145 aminoacids, about 140 amino acids, about 135 amino acids, about 130 aminoacids, about 125 amino acids, about 120 amino acids, about 115 aminoacids, about 110 amino acids, about 105 amino acids, about 100 aminoacids, about 95 amino acids, about 90 amino acids, about 85 amino acids,about 80 amino acids, or about 75 amino acids (inclusive); about 75amino acids to about 800 amino acids, about 780 amino acids, about 760amino acids, about 740 amino acids, about 720 amino acids, about 700amino acids, about 680 amino acids, about 660 amino acids, about 640amino acids, about 620 amino acids, about 600 amino acids, about 580amino acids, about 560 amino acids, about 540 amino acids, about 520amino acids, about 500 amino acids, about 490 amino acids, about 480amino acids, about 470 amino acids, about 460 amino acids, about 450amino acids, about 440 amino acids, about 430 amino acids, about 420amino acids, about 410 amino acids, about 400 amino acids, about 390amino acids, about 380 amino acids, about 370 amino acids, about 360amino acids, about 350 amino acids, about 340 amino acids, about 330amino acids, about 320 amino acids, about 310 amino acids, about 300amino acids, about 290 amino acids, about 280 amino acids, about 270amino acids, about 260 amino acids, about 250 amino acids, about 240amino acids, about 230 amino acids, about 220 amino acids, about 210amino acids, about 200 amino acids, about 195 amino acids, about 190amino acids, about 185 amino acids, about 180 amino acids, about 175amino acids, about 170 amino acids, about 165 amino acids, about 160amino acids, about 155 amino acids, about 150 amino acids, about 145amino acids, about 140 amino acids, about 135 amino acids, about 130amino acids, about 125 amino acids, about 120 amino acids, about 115amino acids, about 110 amino acids, about 105 amino acids, about 100amino acids, about 95 amino acids, about 90 amino acids, about 85 aminoacids, or about 80 amino acids (inclusive); about 80 amino acids toabout 800 amino acids, about 780 amino acids, about 760 amino acids,about 740 amino acids, about 720 amino acids, about 700 amino acids,about 680 amino acids, about 660 amino acids, about 640 amino acids,about 620 amino acids, about 600 amino acids, about 580 amino acids,about 560 amino acids, about 540 amino acids, about 520 amino acids,about 500 amino acids, about 490 amino acids, about 480 amino acids,about 470 amino acids, about 460 amino acids, about 450 amino acids,about 440 amino acids, about 430 amino acids, about 420 amino acids,about 410 amino acids, about 400 amino acids, about 390 amino acids,about 380 amino acids, about 370 amino acids, about 360 amino acids,about 350 amino acids, about 340 amino acids, about 330 amino acids,about 320 amino acids, about 310 amino acids, about 300 amino acids,about 290 amino acids, about 280 amino acids, about 270 amino acids,about 260 amino acids, about 250 amino acids, about 240 amino acids,about 230 amino acids, about 220 amino acids, about 210 amino acids,about 200 amino acids, about 195 amino acids, about 190 amino acids,about 185 amino acids, about 180 amino acids, about 175 amino acids,about 170 amino acids, about 165 amino acids, about 160 amino acids,about 155 amino acids, about 150 amino acids, about 145 amino acids,about 140 amino acids, about 135 amino acids, about 130 amino acids,about 125 amino acids, about 120 amino acids, about 115 amino acids,about 110 amino acids, about 105 amino acids, about 100 amino acids,about 95 amino acids, about 90 amino acids, or about 85 amino acids(inclusive); about 85 amino acids to about 800 amino acids, about 780amino acids, about 760 amino acids, about 740 amino acids, about 720amino acids, about 700 amino acids, about 680 amino acids, about 660amino acids, about 640 amino acids, about 620 amino acids, about 600amino acids, about 580 amino acids, about 560 amino acids, about 540amino acids, about 520 amino acids, about 500 amino acids, about 490amino acids, about 480 amino acids, about 470 amino acids, about 460amino acids, about 450 amino acids, about 440 amino acids, about 430amino acids, about 420 amino acids, about 410 amino acids, about 400amino acids, about 390 amino acids, about 380 amino acids, about 370amino acids, about 360 amino acids, about 350 amino acids, about 340amino acids, about 330 amino acids, about 320 amino acids, about 310amino acids, about 300 amino acids, about 290 amino acids, about 280amino acids, about 270 amino acids, about 260 amino acids, about 250amino acids, about 240 amino acids, about 230 amino acids, about 220amino acids, about 210 amino acids, about 200 amino acids, about 195amino acids, about 190 amino acids, about 185 amino acids, about 180amino acids, about 175 amino acids, about 170 amino acids, about 165amino acids, about 160 amino acids, about 155 amino acids, about 150amino acids, about 145 amino acids, about 140 amino acids, about 135amino acids, about 130 amino acids, about 125 amino acids, about 120amino acids, about 115 amino acids, about 110 amino acids, about 105amino acids, about 100 amino acids, about 95 amino acids, or about 90amino acids (inclusive); about 90 amino acids to about 800 amino acids,about 780 amino acids, about 760 amino acids, about 740 amino acids,about 720 amino acids, about 700 amino acids, about 680 amino acids,about 660 amino acids, about 640 amino acids, about 620 amino acids,about 600 amino acids, about 580 amino acids, about 560 amino acids,about 540 amino acids, about 520 amino acids, about 500 amino acids,about 490 amino acids, about 480 amino acids, about 470 amino acids,about 460 amino acids, about 450 amino acids, about 440 amino acids,about 430 amino acids, about 420 amino acids, about 410 amino acids,about 400 amino acids, about 390 amino acids, about 380 amino acids,about 370 amino acids, about 360 amino acids, about 350 amino acids,about 340 amino acids, about 330 amino acids, about 320 amino acids,about 310 amino acids, about 300 amino acids, about 290 amino acids,about 280 amino acids, about 270 amino acids, about 260 amino acids,about 250 amino acids, about 240 amino acids, about 230 amino acids,about 220 amino acids, about 210 amino acids, about 200 amino acids,about 195 amino acids, about 190 amino acids, about 185 amino acids,about 180 amino acids, about 175 amino acids, about 170 amino acids,about 165 amino acids, about 160 amino acids, about 155 amino acids,about 150 amino acids, about 145 amino acids, about 140 amino acids,about 135 amino acids, about 130 amino acids, about 125 amino acids,about 120 amino acids, about 115 amino acids, about 110 amino acids,about 105 amino acids, about 100 amino acids, or about 95 amino acids(inclusive); about 95 amino acids to about 800 amino acids, about 780amino acids, about 760 amino acids, about 740 amino acids, about 720amino acids, about 700 amino acids, about 680 amino acids, about 660amino acids, about 640 amino acids, about 620 amino acids, about 600amino acids, about 580 amino acids, about 560 amino acids, about 540amino acids, about 520 amino acids, about 500 amino acids, about 490amino acids, about 480 amino acids, about 470 amino acids, about 460amino acids, about 450 amino acids, about 440 amino acids, about 430amino acids, about 420 amino acids, about 410 amino acids, about 400amino acids, about 390 amino acids, about 380 amino acids, about 370amino acids, about 360 amino acids, about 350 amino acids, about 340amino acids, about 330 amino acids, about 320 amino acids, about 310amino acids, about 300 amino acids, about 290 amino acids, about 280amino acids, about 270 amino acids, about 260 amino acids, about 250amino acids, about 240 amino acids, about 230 amino acids, about 220amino acids, about 210 amino acids, about 200 amino acids, about 195amino acids, about 190 amino acids, about 185 amino acids, about 180amino acids, about 175 amino acids, about 170 amino acids, about 165amino acids, about 160 amino acids, about 155 amino acids, about 150amino acids, about 145 amino acids, about 140 amino acids, about 135amino acids, about 130 amino acids, about 125 amino acids, about 120amino acids, about 115 amino acids, about 110 amino acids, about 105amino acids, or about 100 amino acids (inclusive); about 100 amino acidsto about 800 amino acids, about 780 amino acids, about 760 amino acids,about 740 amino acids, about 720 amino acids, about 700 amino acids,about 680 amino acids, about 660 amino acids, about 640 amino acids,about 620 amino acids, about 600 amino acids, about 580 amino acids,about 560 amino acids, about 540 amino acids, about 520 amino acids,about 500 amino acids, about 490 amino acids, about 480 amino acids,about 470 amino acids, about 460 amino acids, about 450 amino acids,about 440 amino acids, about 430 amino acids, about 420 amino acids,about 410 amino acids, about 400 amino acids, about 390 amino acids,about 380 amino acids, about 370 amino acids, about 360 amino acids,about 350 amino acids, about 340 amino acids, about 330 amino acids,about 320 amino acids, about 310 amino acids, about 300 amino acids,about 290 amino acids, about 280 amino acids, about 270 amino acids,about 260 amino acids, about 250 amino acids, about 240 amino acids,about 230 amino acids, about 220 amino acids, about 210 amino acids,about 200 amino acids, about 195 amino acids, about 190 amino acids,about 185 amino acids, about 180 amino acids, about 175 amino acids,about 170 amino acids, about 165 amino acids, about 160 amino acids,about 155 amino acids, about 150 amino acids, about 145 amino acids,about 140 amino acids, about 135 amino acids, about 130 amino acids,about 125 amino acids, about 120 amino acids, about 115 amino acids,about 110 amino acids, or about 105 amino acids (inclusive); about 105amino acids to about 800 amino acids, about 780 amino acids, about 760amino acids, about 740 amino acids, about 720 amino acids, about 700amino acids, about 680 amino acids, about 660 amino acids, about 640amino acids, about 620 amino acids, about 600 amino acids, about 580amino acids, about 560 amino acids, about 540 amino acids, about 520amino acids, about 500 amino acids, about 490 amino acids, about 480amino acids, about 470 amino acids, about 460 amino acids, about 450amino acids, about 440 amino acids, about 430 amino acids, about 420amino acids, about 410 amino acids, about 400 amino acids, about 390amino acids, about 380 amino acids, about 370 amino acids, about 360amino acids, about 350 amino acids, about 340 amino acids, about 330amino acids, about 320 amino acids, about 310 amino acids, about 300amino acids, about 290 amino acids, about 280 amino acids, about 270amino acids, about 260 amino acids, about 250 amino acids, about 240amino acids, about 230 amino acids, about 220 amino acids, about 210amino acids, about 200 amino acids, about 195 amino acids, about 190amino acids, about 185 amino acids, about 180 amino acids, about 175amino acids, about 170 amino acids, about 165 amino acids, about 160amino acids, about 155 amino acids, about 150 amino acids, about 145amino acids, about 140 amino acids, about 135 amino acids, about 130amino acids, about 125 amino acids, about 120 amino acids, about 115amino acids, or about 110 amino acids (inclusive); about 110 amino acidsto about 800 amino acids, about 780 amino acids, about 760 amino acids,about 740 amino acids, about 720 amino acids, about 700 amino acids,about 680 amino acids, about 660 amino acids, about 640 amino acids,about 620 amino acids, about 600 amino acids, about 580 amino acids,about 560 amino acids, about 540 amino acids, about 520 amino acids,about 500 amino acids, about 490 amino acids, about 480 amino acids,about 470 amino acids, about 460 amino acids, about 450 amino acids,about 440 amino acids, about 430 amino acids, about 420 amino acids,about 410 amino acids, about 400 amino acids, about 390 amino acids,about 380 amino acids, about 370 amino acids, about 360 amino acids,about 350 amino acids, about 340 amino acids, about 330 amino acids,about 320 amino acids, about 310 amino acids, about 300 amino acids,about 290 amino acids, about 280 amino acids, about 270 amino acids,about 260 amino acids, about 250 amino acids, about 240 amino acids,about 230 amino acids, about 220 amino acids, about 210 amino acids,about 200 amino acids, about 195 amino acids, about 190 amino acids,about 185 amino acids, about 180 amino acids, about 175 amino acids,about 170 amino acids, about 165 amino acids, about 160 amino acids,about 155 amino acids, about 150 amino acids, about 145 amino acids,about 140 amino acids, about 135 amino acids, about 130 amino acids,about 125 amino acids, about 120 amino acids, or about 115 amino acids(inclusive); about 115 amino acids to about 800 amino acids, about 780amino acids, about 760 amino acids, about 740 amino acids, about 720amino acids, about 700 amino acids, about 680 amino acids, about 660amino acids, about 640 amino acids, about 620 amino acids, about 600amino acids, about 580 amino acids, about 560 amino acids, about 540amino acids, about 520 amino acids, about 500 amino acids, about 490amino acids, about 480 amino acids, about 470 amino acids, about 460amino acids, about 450 amino acids, about 440 amino acids, about 430amino acids, about 420 amino acids, about 410 amino acids, about 400amino acids, about 390 amino acids, about 380 amino acids, about 370amino acids, about 360 amino acids, about 350 amino acids, about 340amino acids, about 330 amino acids, about 320 amino acids, about 310amino acids, about 300 amino acids, about 290 amino acids, about 280amino acids, about 270 amino acids, about 260 amino acids, about 250amino acids, about 240 amino acids, about 230 amino acids, about 220amino acids, about 210 amino acids, about 200 amino acids, about 195amino acids, about 190 amino acids, about 185 amino acids, about 180amino acids, about 175 amino acids, about 170 amino acids, about 165amino acids, about 160 amino acids, about 155 amino acids, about 150amino acids, about 145 amino acids, about 140 amino acids, about 135amino acids, about 130 amino acids, about 125 amino acids, or about 120amino acids (inclusive); about 120 amino acids to about 800 amino acids,about 780 amino acids, about 760 amino acids, about 740 amino acids,about 720 amino acids, about 700 amino acids, about 680 amino acids,about 660 amino acids, about 640 amino acids, about 620 amino acids,about 600 amino acids, about 580 amino acids, about 560 amino acids,about 540 amino acids, about 520 amino acids, about 500 amino acids,about 490 amino acids, about 480 amino acids, about 470 amino acids,about 460 amino acids, about 450 amino acids, about 440 amino acids,about 430 amino acids, about 420 amino acids, about 410 amino acids,about 400 amino acids, about 390 amino acids, about 380 amino acids,about 370 amino acids, about 360 amino acids, about 350 amino acids,about 340 amino acids, about 330 amino acids, about 320 amino acids,about 310 amino acids, about 300 amino acids, about 290 amino acids,about 280 amino acids, about 270 amino acids, about 260 amino acids,about 250 amino acids, about 240 amino acids, about 230 amino acids,about 220 amino acids, about 210 amino acids, about 200 amino acids,about 195 amino acids, about 190 amino acids, about 185 amino acids,about 180 amino acids, about 175 amino acids, about 170 amino acids,about 165 amino acids, about 160 amino acids, about 155 amino acids,about 150 amino acids, about 145 amino acids, about 140 amino acids,about 135 amino acids, about 130 amino acids, or about 125 amino acids(inclusive); about 125 amino acids to about 800 amino acids, about 780amino acids, about 760 amino acids, about 740 amino acids, about 720amino acids, about 700 amino acids, about 680 amino acids, about 660amino acids, about 640 amino acids, about 620 amino acids, about 600amino acids, about 580 amino acids, about 560 amino acids, about 540amino acids, about 520 amino acids, about 500 amino acids, about 490amino acids, about 480 amino acids, about 470 amino acids, about 460amino acids, about 450 amino acids, about 440 amino acids, about 430amino acids, about 420 amino acids, about 410 amino acids, about 400amino acids, about 390 amino acids, about 380 amino acids, about 370amino acids, about 360 amino acids, about 350 amino acids, about 340amino acids, about 330 amino acids, about 320 amino acids, about 310amino acids, about 300 amino acids, about 290 amino acids, about 280amino acids, about 270 amino acids, about 260 amino acids, about 250amino acids, about 240 amino acids, about 230 amino acids, about 220amino acids, about 210 amino acids, about 200 amino acids, about 195amino acids, about 190 amino acids, about 185 amino acids, about 180amino acids, about 175 amino acids, about 170 amino acids, about 165amino acids, about 160 amino acids, about 155 amino acids, about 150amino acids, about 145 amino acids, about 140 amino acids, about 135amino acids, or about 130 amino acids (inclusive); about 130 amino acidsto about 800 amino acids, about 780 amino acids, about 760 amino acids,about 740 amino acids, about 720 amino acids, about 700 amino acids,about 680 amino acids, about 660 amino acids, about 640 amino acids,about 620 amino acids, about 600 amino acids, about 580 amino acids,about 560 amino acids, about 540 amino acids, about 520 amino acids,about 500 amino acids, about 490 amino acids, about 480 amino acids,about 470 amino acids, about 460 amino acids, about 450 amino acids,about 440 amino acids, about 430 amino acids, about 420 amino acids,about 410 amino acids, about 400 amino acids, about 390 amino acids,about 380 amino acids, about 370 amino acids, about 360 amino acids,about 350 amino acids, about 340 amino acids, about 330 amino acids,about 320 amino acids, about 310 amino acids, about 300 amino acids,about 290 amino acids, about 280 amino acids, about 270 amino acids,about 260 amino acids, about 250 amino acids, about 240 amino acids,about 230 amino acids, about 220 amino acids, about 210 amino acids,about 200 amino acids, about 195 amino acids, about 190 amino acids,about 185 amino acids, about 180 amino acids, about 175 amino acids,about 170 amino acids, about 165 amino acids, about 160 amino acids,about 155 amino acids, about 150 amino acids, about 145 amino acids,about 140 amino acids, or about 135 amino acids (inclusive); about 135amino acids to about 800 amino acids, about 780 amino acids, about 760amino acids, about 740 amino acids, about 720 amino acids, about 700amino acids, about 680 amino acids, about 660 amino acids, about 640amino acids, about 620 amino acids, about 600 amino acids, about 580amino acids, about 560 amino acids, about 540 amino acids, about 520amino acids, about 500 amino acids, about 490 amino acids, about 480amino acids, about 470 amino acids, about 460 amino acids, about 450amino acids, about 440 amino acids, about 430 amino acids, about 420amino acids, about 410 amino acids, about 400 amino acids, about 390amino acids, about 380 amino acids, about 370 amino acids, about 360amino acids, about 350 amino acids, about 340 amino acids, about 330amino acids, about 320 amino acids, about 310 amino acids, about 300amino acids, about 290 amino acids, about 280 amino acids, about 270amino acids, about 260 amino acids, about 250 amino acids, about 240amino acids, about 230 amino acids, about 220 amino acids, about 210amino acids, about 200 amino acids, about 195 amino acids, about 190amino acids, about 185 amino acids, about 180 amino acids, about 175amino acids, about 170 amino acids, about 165 amino acids, about 160amino acids, about 155 amino acids, about 150 amino acids, about 145amino acids, or about 140 amino acids (inclusive); about 140 amino acidsto about 800 amino acids, about 780 amino acids, about 760 amino acids,about 740 amino acids, about 720 amino acids, about 700 amino acids,about 680 amino acids, about 660 amino acids, about 640 amino acids,about 620 amino acids, about 600 amino acids, about 580 amino acids,about 560 amino acids, about 540 amino acids, about 520 amino acids,about 500 amino acids, about 490 amino acids, about 480 amino acids,about 470 amino acids, about 460 amino acids, about 450 amino acids,about 440 amino acids, about 430 amino acids, about 420 amino acids,about 410 amino acids, about 400 amino acids, about 390 amino acids,about 380 amino acids, about 370 amino acids, about 360 amino acids,about 350 amino acids, about 340 amino acids, about 330 amino acids,about 320 amino acids, about 310 amino acids, about 300 amino acids,about 290 amino acids, about 280 amino acids, about 270 amino acids,about 260 amino acids, about 250 amino acids, about 240 amino acids,about 230 amino acids, about 220 amino acids, about 210 amino acids,about 200 amino acids, about 195 amino acids, about 190 amino acids,about 185 amino acids, about 180 amino acids, about 175 amino acids,about 170 amino acids, about 165 amino acids, about 160 amino acids,about 155 amino acids, about 150 amino acids, or about 145 amino acids(inclusive); about 145 amino acids to about 800 amino acids, about 780amino acids, about 760 amino acids, about 740 amino acids, about 720amino acids, about 700 amino acids, about 680 amino acids, about 660amino acids, about 640 amino acids, about 620 amino acids, about 600amino acids, about 580 amino acids, about 560 amino acids, about 540amino acids, about 520 amino acids, about 500 amino acids, about 490amino acids, about 480 amino acids, about 470 amino acids, about 460amino acids, about 450 amino acids, about 440 amino acids, about 430amino acids, about 420 amino acids, about 410 amino acids, about 400amino acids, about 390 amino acids, about 380 amino acids, about 370amino acids, about 360 amino acids, about 350 amino acids, about 340amino acids, about 330 amino acids, about 320 amino acids, about 310amino acids, about 300 amino acids, about 290 amino acids, about 280amino acids, about 270 amino acids, about 260 amino acids, about 250amino acids, about 240 amino acids, about 230 amino acids, about 220amino acids, about 210 amino acids, about 200 amino acids, about 195amino acids, about 190 amino acids, about 185 amino acids, about 180amino acids, about 175 amino acids, about 170 amino acids, about 165amino acids, about 160 amino acids, about 155 amino acids, or about 150amino acids (inclusive); about 150 amino acids to about 800 amino acids,about 780 amino acids, about 760 amino acids, about 740 amino acids,about 720 amino acids, about 700 amino acids, about 680 amino acids,about 660 amino acids, about 640 amino acids, about 620 amino acids,about 600 amino acids, about 580 amino acids, about 560 amino acids,about 540 amino acids, about 520 amino acids, about 500 amino acids,about 490 amino acids, about 480 amino acids, about 470 amino acids,about 460 amino acids, about 450 amino acids, about 440 amino acids,about 430 amino acids, about 420 amino acids, about 410 amino acids,about 400 amino acids, about 390 amino acids, about 380 amino acids,about 370 amino acids, about 360 amino acids, about 350 amino acids,about 340 amino acids, about 330 amino acids, about 320 amino acids,about 310 amino acids, about 300 amino acids, about 290 amino acids,about 280 amino acids, about 270 amino acids, about 260 amino acids,about 250 amino acids, about 240 amino acids, about 230 amino acids,about 220 amino acids, about 210 amino acids, about 200 amino acids,about 195 amino acids, about 190 amino acids, about 185 amino acids,about 180 amino acids, about 175 amino acids, about 170 amino acids,about 165 amino acids, about 160 amino acids, or about 155 amino acids(inclusive); about 155 amino acids to about 800 amino acids, about 780amino acids, about 760 amino acids, about 740 amino acids, about 720amino acids, about 700 amino acids, about 680 amino acids, about 660amino acids, about 640 amino acids, about 620 amino acids, about 600amino acids, about 580 amino acids, about 560 amino acids, about 540amino acids, about 520 amino acids, about 500 amino acids, about 490amino acids, about 480 amino acids, about 470 amino acids, about 460amino acids, about 450 amino acids, about 440 amino acids, about 430amino acids, about 420 amino acids, about 410 amino acids, about 400amino acids, about 390 amino acids, about 380 amino acids, about 370amino acids, about 360 amino acids, about 350 amino acids, about 340amino acids, about 330 amino acids, about 320 amino acids, about 310amino acids, about 300 amino acids, about 290 amino acids, about 280amino acids, about 270 amino acids, about 260 amino acids, about 250amino acids, about 240 amino acids, about 230 amino acids, about 220amino acids, about 210 amino acids, about 200 amino acids, about 195amino acids, about 190 amino acids, about 185 amino acids, about 180amino acids, about 175 amino acids, about 170 amino acids, about 165amino acids, or about 160 amino acids (inclusive); about 160 amino acidsto about 800 amino acids, about 780 amino acids, about 760 amino acids,about 740 amino acids, about 720 amino acids, about 700 amino acids,about 680 amino acids, about 660 amino acids, about 640 amino acids,about 620 amino acids, about 600 amino acids, about 580 amino acids,about 560 amino acids, about 540 amino acids, about 520 amino acids,about 500 amino acids, about 490 amino acids, about 480 amino acids,about 470 amino acids, about 460 amino acids, about 450 amino acids,about 440 amino acids, about 430 amino acids, about 420 amino acids,about 410 amino acids, about 400 amino acids, about 390 amino acids,about 380 amino acids, about 370 amino acids, about 360 amino acids,about 350 amino acids, about 340 amino acids, about 330 amino acids,about 320 amino acids, about 310 amino acids, about 300 amino acids,about 290 amino acids, about 280 amino acids, about 270 amino acids,about 260 amino acids, about 250 amino acids, about 240 amino acids,about 230 amino acids, about 220 amino acids, about 210 amino acids,about 200 amino acids, about 195 amino acids, about 190 amino acids,about 185 amino acids, about 180 amino acids, about 175 amino acids,about 170 amino acids, or about 165 amino acids (inclusive); about 165amino acids to about 800 amino acids, about 780 amino acids, about 760amino acids, about 740 amino acids, about 720 amino acids, about 700amino acids, about 680 amino acids, about 660 amino acids, about 640amino acids, about 620 amino acids, about 600 amino acids, about 580amino acids, about 560 amino acids, about 540 amino acids, about 520amino acids, about 500 amino acids, about 490 amino acids, about 480amino acids, about 470 amino acids, about 460 amino acids, about 450amino acids, about 440 amino acids, about 430 amino acids, about 420amino acids, about 410 amino acids, about 400 amino acids, about 390amino acids, about 380 amino acids, about 370 amino acids, about 360amino acids, about 350 amino acids, about 340 amino acids, about 330amino acids, about 320 amino acids, about 310 amino acids, about 300amino acids, about 290 amino acids, about 280 amino acids, about 270amino acids, about 260 amino acids, about 250 amino acids, about 240amino acids, about 230 amino acids, about 220 amino acids, about 210amino acids, about 200 amino acids, about 195 amino acids, about 190amino acids, about 185 amino acids, about 180 amino acids, about 175amino acids, or about 170 amino acids (inclusive); about 170 amino acidsto about 800 amino acids, about 780 amino acids, about 760 amino acids,about 740 amino acids, about 720 amino acids, about 700 amino acids,about 680 amino acids, about 660 amino acids, about 640 amino acids,about 620 amino acids, about 600 amino acids, about 580 amino acids,about 560 amino acids, about 540 amino acids, about 520 amino acids,about 500 amino acids, about 490 amino acids, about 480 amino acids,about 470 amino acids, about 460 amino acids, about 450 amino acids,about 440 amino acids, about 430 amino acids, about 420 amino acids,about 410 amino acids, about 400 amino acids, about 390 amino acids,about 380 amino acids, about 370 amino acids, about 360 amino acids,about 350 amino acids, about 340 amino acids, about 330 amino acids,about 320 amino acids, about 310 amino acids, about 300 amino acids,about 290 amino acids, about 280 amino acids, about 270 amino acids,about 260 amino acids, about 250 amino acids, about 240 amino acids,about 230 amino acids, about 220 amino acids, about 210 amino acids,about 200 amino acids, about 195 amino acids, about 190 amino acids,about 185 amino acids, about 180 amino acids, or about 175 amino acids(inclusive); about 175 amino acids to about 800 amino acids, about 780amino acids, about 760 amino acids, about 740 amino acids, about 720amino acids, about 700 amino acids, about 680 amino acids, about 660amino acids, about 640 amino acids, about 620 amino acids, about 600amino acids, about 580 amino acids, about 560 amino acids, about 540amino acids, about 520 amino acids, about 500 amino acids, about 490amino acids, about 480 amino acids, about 470 amino acids, about 460amino acids, about 450 amino acids, about 440 amino acids, about 430amino acids, about 420 amino acids, about 410 amino acids, about 400amino acids, about 390 amino acids, about 380 amino acids, about 370amino acids, about 360 amino acids, about 350 amino acids, about 340amino acids, about 330 amino acids, about 320 amino acids, about 310amino acids, about 300 amino acids, about 290 amino acids, about 280amino acids, about 270 amino acids, about 260 amino acids, about 250amino acids, about 240 amino acids, about 230 amino acids, about 220amino acids, about 210 amino acids, about 200 amino acids, about 195amino acids, about 190 amino acids, about 185 amino acids, or about 180amino acids (inclusive); about 180 amino acids to about 800 amino acids,about 780 amino acids, about 760 amino acids, about 740 amino acids,about 720 amino acids, about 700 amino acids, about 680 amino acids,about 660 amino acids, about 640 amino acids, about 620 amino acids,about 600 amino acids, about 580 amino acids, about 560 amino acids,about 540 amino acids, about 520 amino acids, about 500 amino acids,about 490 amino acids, about 480 amino acids, about 470 amino acids,about 460 amino acids, about 450 amino acids, about 440 amino acids,about 430 amino acids, about 420 amino acids, about 410 amino acids,about 400 amino acids, about 390 amino acids, about 380 amino acids,about 370 amino acids, about 360 amino acids, about 350 amino acids,about 340 amino acids, about 330 amino acids, about 320 amino acids,about 310 amino acids, about 300 amino acids, about 290 amino acids,about 280 amino acids, about 270 amino acids, about 260 amino acids,about 250 amino acids, about 240 amino acids, about 230 amino acids,about 220 amino acids, about 210 amino acids, about 200 amino acids,about 195 amino acids, about 190 amino acids, or about 185 amino acids(inclusive); about 185 amino acids to about 800 amino acids, about 780amino acids, about 760 amino acids, about 740 amino acids, about 720amino acids, about 700 amino acids, about 680 amino acids, about 660amino acids, about 640 amino acids, about 620 amino acids, about 600amino acids, about 580 amino acids, about 560 amino acids, about 540amino acids, about 520 amino acids, about 500 amino acids, about 490amino acids, about 480 amino acids, about 470 amino acids, about 460amino acids, about 450 amino acids, about 440 amino acids, about 430amino acids, about 420 amino acids, about 410 amino acids, about 400amino acids, about 390 amino acids, about 380 amino acids, about 370amino acids, about 360 amino acids, about 350 amino acids, about 340amino acids, about 330 amino acids, about 320 amino acids, about 310amino acids, about 300 amino acids, about 290 amino acids, about 280amino acids, about 270 amino acids, about 260 amino acids, about 250amino acids, about 240 amino acids, about 230 amino acids, about 220amino acids, about 210 amino acids, about 200 amino acids, about 195amino acids, or about 190 amino acids (inclusive); about 190 amino acidsto about 800 amino acids, about 780 amino acids, about 760 amino acids,about 740 amino acids, about 720 amino acids, about 700 amino acids,about 680 amino acids, about 660 amino acids, about 640 amino acids,about 620 amino acids, about 600 amino acids, about 580 amino acids,about 560 amino acids, about 540 amino acids, about 520 amino acids,about 500 amino acids, about 490 amino acids, about 480 amino acids,about 470 amino acids, about 460 amino acids, about 450 amino acids,about 440 amino acids, about 430 amino acids, about 420 amino acids,about 410 amino acids, about 400 amino acids, about 390 amino acids,about 380 amino acids, about 370 amino acids, about 360 amino acids,about 350 amino acids, about 340 amino acids, about 330 amino acids,about 320 amino acids, about 310 amino acids, about 300 amino acids,about 290 amino acids, about 280 amino acids, about 270 amino acids,about 260 amino acids, about 250 amino acids, about 240 amino acids,about 230 amino acids, about 220 amino acids, about 210 amino acids,about 200 amino acids, or about 195 amino acids (inclusive); about 195amino acids to about 800 amino acids, about 780 amino acids, about 760amino acids, about 740 amino acids, about 720 amino acids, about 700amino acids, about 680 amino acids, about 660 amino acids, about 640amino acids, about 620 amino acids, about 600 amino acids, about 580amino acids, about 560 amino acids, about 540 amino acids, about 520amino acids, about 500 amino acids, about 490 amino acids, about 480amino acids, about 470 amino acids, about 460 amino acids, about 450amino acids, about 440 amino acids, about 430 amino acids, about 420amino acids, about 410 amino acids, about 400 amino acids, about 390amino acids, about 380 amino acids, about 370 amino acids, about 360amino acids, about 350 amino acids, about 340 amino acids, about 330amino acids, about 320 amino acids, about 310 amino acids, about 300amino acids, about 290 amino acids, about 280 amino acids, about 270amino acids, about 260 amino acids, about 250 amino acids, about 240amino acids, about 230 amino acids, about 220 amino acids, about 210amino acids, or about 200 amino acids (inclusive); about 200 amino acidsto about 800 amino acids, about 780 amino acids, about 760 amino acids,about 740 amino acids, about 720 amino acids, about 700 amino acids,about 680 amino acids, about 660 amino acids, about 640 amino acids,about 620 amino acids, about 600 amino acids, about 580 amino acids,about 560 amino acids, about 540 amino acids, about 520 amino acids,about 500 amino acids, about 490 amino acids, about 480 amino acids,about 470 amino acids, about 460 amino acids, about 450 amino acids,about 440 amino acids, about 430 amino acids, about 420 amino acids,about 410 amino acids, about 400 amino acids, about 390 amino acids,about 380 amino acids, about 370 amino acids, about 360 amino acids,about 350 amino acids, about 340 amino acids, about 330 amino acids,about 320 amino acids, about 310 amino acids, about 300 amino acids,about 290 amino acids, about 280 amino acids, about 270 amino acids,about 260 amino acids, about 250 amino acids, about 240 amino acids,about 230 amino acids, about 220 amino acids, or about 210 amino acids(inclusive); about 210 amino acids to about 800 amino acids, about 780amino acids, about 760 amino acids, about 740 amino acids, about 720amino acids, about 700 amino acids, about 680 amino acids, about 660amino acids, about 640 amino acids, about 620 amino acids, about 600amino acids, about 580 amino acids, about 560 amino acids, about 540amino acids, about 520 amino acids, about 500 amino acids, about 490amino acids, about 480 amino acids, about 470 amino acids, about 460amino acids, about 450 amino acids, about 440 amino acids, about 430amino acids, about 420 amino acids, about 410 amino acids, about 400amino acids, about 390 amino acids, about 380 amino acids, about 370amino acids, about 360 amino acids, about 350 amino acids, about 340amino acids, about 330 amino acids, about 320 amino acids, about 310amino acids, about 300 amino acids, about 290 amino acids, about 280amino acids, about 270 amino acids, about 260 amino acids, about 250amino acids, about 240 amino acids, about 230 amino acids, or about 220amino acids (inclusive); about 220 amino acids to about 800 amino acids,about 780 amino acids, about 760 amino acids, about 740 amino acids,about 720 amino acids, about 700 amino acids, about 680 amino acids,about 660 amino acids, about 640 amino acids, about 620 amino acids,about 600 amino acids, about 580 amino acids, about 560 amino acids,about 540 amino acids, about 520 amino acids, about 500 amino acids,about 490 amino acids, about 480 amino acids, about 470 amino acids,about 460 amino acids, about 450 amino acids, about 440 amino acids,about 430 amino acids, about 420 amino acids, about 410 amino acids,about 400 amino acids, about 390 amino acids, about 380 amino acids,about 370 amino acids, about 360 amino acids, about 350 amino acids,about 340 amino acids, about 330 amino acids, about 320 amino acids,about 310 amino acids, about 300 amino acids, about 290 amino acids,about 280 amino acids, about 270 amino acids, about 260 amino acids,about 250 amino acids, about 240 amino acids, or about 230 amino acids(inclusive); about 230 amino acids to about 800 amino acids, about 780amino acids, about 760 amino acids, about 740 amino acids, about 720amino acids, about 700 amino acids, about 680 amino acids, about 660amino acids, about 640 amino acids, about 620 amino acids, about 600amino acids, about 580 amino acids, about 560 amino acids, about 540amino acids, about 520 amino acids, about 500 amino acids, about 490amino acids, about 480 amino acids, about 470 amino acids, about 460amino acids, about 450 amino acids, about 440 amino acids, about 430amino acids, about 420 amino acids, about 410 amino acids, about 400amino acids, about 390 amino acids, about 380 amino acids, about 370amino acids, about 360 amino acids, about 350 amino acids, about 340amino acids, about 330 amino acids, about 320 amino acids, about 310amino acids, about 300 amino acids, about 290 amino acids, about 280amino acids, about 270 amino acids, about 260 amino acids, about 250amino acids, or about 240 amino acids (inclusive); about 240 amino acidsto about 800 amino acids, about 780 amino acids, about 760 amino acids,about 740 amino acids, about 720 amino acids, about 700 amino acids,about 680 amino acids, about 660 amino acids, about 640 amino acids,about 620 amino acids, about 600 amino acids, about 580 amino acids,about 560 amino acids, about 540 amino acids, about 520 amino acids,about 500 amino acids, about 490 amino acids, about 480 amino acids,about 470 amino acids, about 460 amino acids, about 450 amino acids,about 440 amino acids, about 430 amino acids, about 420 amino acids,about 410 amino acids, about 400 amino acids, about 390 amino acids,about 380 amino acids, about 370 amino acids, about 360 amino acids,about 350 amino acids, about 340 amino acids, about 330 amino acids,about 320 amino acids, about 310 amino acids, about 300 amino acids,about 290 amino acids, about 280 amino acids, about 270 amino acids,about 260 amino acids, or about 250 amino acids (inclusive); about 250amino acids to about 800 amino acids, about 780 amino acids, about 760amino acids, about 740 amino acids, about 720 amino acids, about 700amino acids, about 680 amino acids, about 660 amino acids, about 640amino acids, about 620 amino acids, about 600 amino acids, about 580amino acids, about 560 amino acids, about 540 amino acids, about 520amino acids, about 500 amino acids, about 490 amino acids, about 480amino acids, about 470 amino acids, about 460 amino acids, about 450amino acids, about 440 amino acids, about 430 amino acids, about 420amino acids, about 410 amino acids, about 400 amino acids, about 390amino acids, about 380 amino acids, about 370 amino acids, about 360amino acids, about 350 amino acids, about 340 amino acids, about 330amino acids, about 320 amino acids, about 310 amino acids, about 300amino acids, about 290 amino acids, about 280 amino acids, about 270amino acids, or about 260 amino acids (inclusive); about 260 amino acidsto about 800 amino acids, about 780 amino acids, about 760 amino acids,about 740 amino acids, about 720 amino acids, about 700 amino acids,about 680 amino acids, about 660 amino acids, about 640 amino acids,about 620 amino acids, about 600 amino acids, about 580 amino acids,about 560 amino acids, about 540 amino acids, about 520 amino acids,about 500 amino acids, about 490 amino acids, about 480 amino acids,about 470 amino acids, about 460 amino acids, about 450 amino acids,about 440 amino acids, about 430 amino acids, about 420 amino acids,about 410 amino acids, about 400 amino acids, about 390 amino acids,about 380 amino acids, about 370 amino acids, about 360 amino acids,about 350 amino acids, about 340 amino acids, about 330 amino acids,about 320 amino acids, about 310 amino acids, about 300 amino acids,about 290 amino acids, about 280 amino acids, or about 270 amino acids(inclusive); about 270 amino acids to about 800 amino acids, about 780amino acids, about 760 amino acids, about 740 amino acids, about 720amino acids, about 700 amino acids, about 680 amino acids, about 660amino acids, about 640 amino acids, about 620 amino acids, about 600amino acids, about 580 amino acids, about 560 amino acids, about 540amino acids, about 520 amino acids, about 500 amino acids, about 490amino acids, about 480 amino acids, about 470 amino acids, about 460amino acids, about 450 amino acids, about 440 amino acids, about 430amino acids, about 420 amino acids, about 410 amino acids, about 400amino acids, about 390 amino acids, about 380 amino acids, about 370amino acids, about 360 amino acids, about 350 amino acids, about 340amino acids, about 330 amino acids, about 320 amino acids, about 310amino acids, about 300 amino acids, about 290 amino acids, or about 280amino acids (inclusive); about 280 amino acids to about 800 amino acids,about 780 amino acids, about 760 amino acids, about 740 amino acids,about 720 amino acids, about 700 amino acids, about 680 amino acids,about 660 amino acids, about 640 amino acids, about 620 amino acids,about 600 amino acids, about 580 amino acids, about 560 amino acids,about 540 amino acids, about 520 amino acids, about 500 amino acids,about 490 amino acids, about 480 amino acids, about 470 amino acids,about 460 amino acids, about 450 amino acids, about 440 amino acids,about 430 amino acids, about 420 amino acids, about 410 amino acids,about 400 amino acids, about 390 amino acids, about 380 amino acids,about 370 amino acids, about 360 amino acids, about 350 amino acids,about 340 amino acids, about 330 amino acids, about 320 amino acids,about 310 amino acids, about 300 amino acids, or about 290 amino acids(inclusive); about 290 amino acids to about 800 amino acids, about 780amino acids, about 760 amino acids, about 740 amino acids, about 720amino acids, about 700 amino acids, about 680 amino acids, about 660amino acids, about 640 amino acids, about 620 amino acids, about 600amino acids, about 580 amino acids, about 560 amino acids, about 540amino acids, about 520 amino acids, about 500 amino acids, about 490amino acids, about 480 amino acids, about 470 amino acids, about 460amino acids, about 450 amino acids, about 440 amino acids, about 430amino acids, about 420 amino acids, about 410 amino acids, about 400amino acids, about 390 amino acids, about 380 amino acids, about 370amino acids, about 360 amino acids, about 350 amino acids, about 340amino acids, about 330 amino acids, about 320 amino acids, about 310amino acids, or about 300 amino acids (inclusive); about 300 amino acidsto about 800 amino acids, about 780 amino acids, about 760 amino acids,about 740 amino acids, about 720 amino acids, about 700 amino acids,about 680 amino acids, about 660 amino acids, about 640 amino acids,about 620 amino acids, about 600 amino acids, about 580 amino acids,about 560 amino acids, about 540 amino acids, about 520 amino acids,about 500 amino acids, about 490 amino acids, about 480 amino acids,about 470 amino acids, about 460 amino acids, about 450 amino acids,about 440 amino acids, about 430 amino acids, about 420 amino acids,about 410 amino acids, about 400 amino acids, about 390 amino acids,about 380 amino acids, about 370 amino acids, about 360 amino acids,about 350 amino acids, about 340 amino acids, about 330 amino acids,about 320 amino acids, about 310 amino acids, about 300 amino acids, orabout 290 amino acids (inclusive); about 290 amino acids to about 800amino acids, about 780 amino acids, about 760 amino acids, about 740amino acids, about 720 amino acids, about 700 amino acids, about 680amino acids, about 660 amino acids, about 640 amino acids, about 620amino acids, about 600 amino acids, about 580 amino acids, about 560amino acids, about 540 amino acids, about 520 amino acids, about 500amino acids, about 490 amino acids, about 480 amino acids, about 470amino acids, about 460 amino acids, about 450 amino acids, about 440amino acids, about 430 amino acids, about 420 amino acids, about 410amino acids, about 400 amino acids, about 390 amino acids, about 380amino acids, about 370 amino acids, about 360 amino acids, about 350amino acids, about 340 amino acids, about 330 amino acids, about 320amino acids, about 310 amino acids, or about 300 amino acids(inclusive); about 300 amino acids to about 800 amino acids, about 780amino acids, about 760 amino acids, about 740 amino acids, about 720amino acids, about 700 amino acids, about 680 amino acids, about 660amino acids, about 640 amino acids, about 620 amino acids, about 600amino acids, about 580 amino acids, about 560 amino acids, about 540amino acids, about 520 amino acids, about 500 amino acids, about 490amino acids, about 480 amino acids, about 470 amino acids, about 460amino acids, about 450 amino acids, about 440 amino acids, about 430amino acids, about 420 amino acids, about 410 amino acids, about 400amino acids, about 390 amino acids, about 380 amino acids, about 370amino acids, about 360 amino acids, about 350 amino acids, about 340amino acids, about 330 amino acids, about 320 amino acids, or about 310amino acids (inclusive); about 310 amino acids to about 800 amino acids,about 780 amino acids, about 760 amino acids, about 740 amino acids,about 720 amino acids, about 700 amino acids, about 680 amino acids,about 660 amino acids, about 640 amino acids, about 620 amino acids,about 600 amino acids, about 580 amino acids, about 560 amino acids,about 540 amino acids, about 520 amino acids, about 500 amino acids,about 490 amino acids, about 480 amino acids, about 470 amino acids,about 460 amino acids, about 450 amino acids, about 440 amino acids,about 430 amino acids, about 420 amino acids, about 410 amino acids,about 400 amino acids, about 390 amino acids, about 380 amino acids,about 370 amino acids, about 360 amino acids, about 350 amino acids,about 340 amino acids, about 330 amino acids, or about 320 amino acids(inclusive); about 320 amino acids to about 800 amino acids, about 780amino acids, about 760 amino acids, about 740 amino acids, about 720amino acids, about 700 amino acids, about 680 amino acids, about 660amino acids, about 640 amino acids, about 620 amino acids, about 600amino acids, about 580 amino acids, about 560 amino acids, about 540amino acids, about 520 amino acids, about 500 amino acids, about 490amino acids, about 480 amino acids, about 470 amino acids, about 460amino acids, about 450 amino acids, about 440 amino acids, about 430amino acids, about 420 amino acids, about 410 amino acids, about 400amino acids, about 390 amino acids, about 380 amino acids, about 370amino acids, about 360 amino acids, about 350 amino acids, about 340amino acids, or about 330 amino acids (inclusive); about 330 amino acidsto about 800 amino acids, about 780 amino acids, about 760 amino acids,about 740 amino acids, about 720 amino acids, about 700 amino acids,about 680 amino acids, about 660 amino acids, about 640 amino acids,about 620 amino acids, about 600 amino acids, about 580 amino acids,about 560 amino acids, about 540 amino acids, about 520 amino acids,about 500 amino acids, about 490 amino acids, about 480 amino acids,about 470 amino acids, about 460 amino acids, about 450 amino acids,about 440 amino acids, about 430 amino acids, about 420 amino acids,about 410 amino acids, about 400 amino acids, about 390 amino acids,about 380 amino acids, about 370 amino acids, about 360 amino acids,about 350 amino acids, or about 340 amino acids (inclusive); about 340amino acids to about 800 amino acids, about 780 amino acids, about 760amino acids, about 740 amino acids, about 720 amino acids, about 700amino acids, about 680 amino acids, about 660 amino acids, about 640amino acids, about 620 amino acids, about 600 amino acids, about 580amino acids, about 560 amino acids, about 540 amino acids, about 520amino acids, about 500 amino acids, about 490 amino acids, about 480amino acids, about 470 amino acids, about 460 amino acids, about 450amino acids, about 440 amino acids, about 430 amino acids, about 420amino acids, about 410 amino acids, about 400 amino acids, about 390amino acids, about 380 amino acids, about 370 amino acids, about 360amino acids, or about 350 amino acids (inclusive); about 350 amino acidsto about 800 amino acids, about 780 amino acids, about 760 amino acids,about 740 amino acids, about 720 amino acids, about 700 amino acids,about 680 amino acids, about 660 amino acids, about 640 amino acids,about 620 amino acids, about 600 amino acids, about 580 amino acids,about 560 amino acids, about 540 amino acids, about 520 amino acids,about 500 amino acids, about 490 amino acids, about 480 amino acids,about 470 amino acids, about 460 amino acids, about 450 amino acids,about 440 amino acids, about 430 amino acids, about 420 amino acids,about 410 amino acids, about 400 amino acids, about 390 amino acids,about 380 amino acids, about 370 amino acids, or about 360 amino acids(inclusive); about 360 amino acids to about 800 amino acids, about 780amino acids, about 760 amino acids, about 740 amino acids, about 720amino acids, about 700 amino acids, about 680 amino acids, about 660amino acids, about 640 amino acids, about 620 amino acids, about 600amino acids, about 580 amino acids, about 560 amino acids, about 540amino acids, about 520 amino acids, about 500 amino acids, about 490amino acids, about 480 amino acids, about 470 amino acids, about 460amino acids, about 450 amino acids, about 440 amino acids, about 430amino acids, about 420 amino acids, about 410 amino acids, about 400amino acids, about 390 amino acids, about 380 amino acids, about 370amino acids (inclusive); about 370 amino acids to about 800 amino acids,about 780 amino acids, about 760 amino acids, about 740 amino acids,about 720 amino acids, about 700 amino acids, about 680 amino acids,about 660 amino acids, about 640 amino acids, about 620 amino acids,about 600 amino acids, about 580 amino acids, about 560 amino acids,about 540 amino acids, about 520 amino acids, about 500 amino acids,about 490 amino acids, about 480 amino acids, about 470 amino acids,about 460 amino acids, about 450 amino acids, about 440 amino acids,about 430 amino acids, about 420 amino acids, about 410 amino acids,about 400 amino acids, about 390 amino acids, or about 380 amino acids(inclusive); about 380 amino acids to about 800 amino acids, about 780amino acids, about 760 amino acids, about 740 amino acids, about 720amino acids, about 700 amino acids, about 680 amino acids, about 660amino acids, about 640 amino acids, about 620 amino acids, about 600amino acids, about 580 amino acids, about 560 amino acids, about 540amino acids, about 520 amino acids, about 500 amino acids, about 490amino acids, about 480 amino acids, about 470 amino acids, about 460amino acids, about 450 amino acids, about 440 amino acids, about 430amino acids, about 420 amino acids, about 410 amino acids, about 400amino acids, or about 390 amino acids (inclusive); about 390 amino acidsto about 800 amino acids, about 780 amino acids, about 760 amino acids,about 740 amino acids, about 720 amino acids, about 700 amino acids,about 680 amino acids, about 660 amino acids, about 640 amino acids,about 620 amino acids, about 600 amino acids, about 580 amino acids,about 560 amino acids, about 540 amino acids, about 520 amino acids,about 500 amino acids, about 490 amino acids, about 480 amino acids,about 470 amino acids, about 460 amino acids, about 450 amino acids,about 440 amino acids, about 430 amino acids, about 420 amino acids,about 410 amino acids, or about 400 amino acids (inclusive); about 400amino acids to about 800 amino acids, about 780 amino acids, about 760amino acids, about 740 amino acids, about 720 amino acids, about 700amino acids, about 680 amino acids, about 660 amino acids, about 640amino acids, about 620 amino acids, about 600 amino acids, about 580amino acids, about 560 amino acids, about 540 amino acids, about 520amino acids, about 500 amino acids, about 490 amino acids, about 480amino acids, about 470 amino acids, about 460 amino acids, about 450amino acids, about 440 amino acids, about 430 amino acids, about 420amino acids, or about 410 amino acids (inclusive); about 410 amino acidsto about 800 amino acids, about 780 amino acids, about 760 amino acids,about 740 amino acids, about 720 amino acids, about 700 amino acids,about 680 amino acids, about 660 amino acids, about 640 amino acids,about 620 amino acids, about 600 amino acids, about 580 amino acids,about 560 amino acids, about 540 amino acids, about 520 amino acids,about 500 amino acids, about 490 amino acids, about 480 amino acids,about 470 amino acids, about 460 amino acids, about 450 amino acids,about 440 amino acids, about 430 amino acids, or about 420 amino acids(inclusive); about 420 amino acids to about 800 amino acids, about 780amino acids, about 760 amino acids, about 740 amino acids, about 720amino acids, about 700 amino acids, about 680 amino acids, about 660amino acids, about 640 amino acids, about 620 amino acids, about 600amino acids, about 580 amino acids, about 560 amino acids, about 540amino acids, about 520 amino acids, about 500 amino acids, about 490amino acids, about 480 amino acids, about 470 amino acids, about 460amino acids, about 450 amino acids, or about 440 amino acids(inclusive); about 430 amino acids to about 800 amino acids, about 780amino acids, about 760 amino acids, about 740 amino acids, about 720amino acids, about 700 amino acids, about 680 amino acids, about 660amino acids, about 640 amino acids, about 620 amino acids, about 600amino acids, about 580 amino acids, about 560 amino acids, about 540amino acids, about 520 amino acids, about 500 amino acids, about 490amino acids, about 480 amino acids, about 470 amino acids, about 460amino acids, about 450 amino acids, or about 440 amino acids(inclusive); about 440 amino acids to about 800 amino acids, about 780amino acids, about 760 amino acids, about 740 amino acids, about 720amino acids, about 700 amino acids, about 680 amino acids, about 660amino acids, about 640 amino acids, about 620 amino acids, about 600amino acids, about 580 amino acids, about 560 amino acids, about 540amino acids, about 520 amino acids, about 500 amino acids, about 490amino acids, about 480 amino acids, about 470 amino acids, about 460amino acids, or about 450 amino acids (inclusive); about 450 amino acidsto about 800 amino acids, about 780 amino acids, about 760 amino acids,about 740 amino acids, about 720 amino acids, about 700 amino acids,about 680 amino acids, about 660 amino acids, about 640 amino acids,about 620 amino acids, about 600 amino acids, about 580 amino acids,about 560 amino acids, about 540 amino acids, about 520 amino acids,about 500 amino acids, about 490 amino acids, about 480 amino acids,about 470 amino acids, or about 460 amino acids (inclusive); about 460amino acids to about 800 amino acids, about 780 amino acids, about 760amino acids, about 740 amino acids, about 720 amino acids, about 700amino acids, about 680 amino acids, about 660 amino acids, about 640amino acids, about 620 amino acids, about 600 amino acids, about 580amino acids, about 560 amino acids, about 540 amino acids, about 520amino acids, about 500 amino acids, about 490 amino acids, about 480amino acids, about 470 amino acids (inclusive); about 470 amino acids toabout 800 amino acids, about 780 amino acids, about 760 amino acids,about 740 amino acids, about 720 amino acids, about 700 amino acids,about 680 amino acids, about 660 amino acids, about 640 amino acids,about 620 amino acids, about 600 amino acids, about 580 amino acids,about 560 amino acids, about 540 amino acids, about 520 amino acids,about 500 amino acids, about 490 amino acids, or about 480 amino acids(inclusive); about 480 amino acids to about 800 amino acids, about 780amino acids, about 760 amino acids, about 740 amino acids, about 720amino acids, about 700 amino acids, about 680 amino acids, about 660amino acids, about 640 amino acids, about 620 amino acids, about 600amino acids, about 580 amino acids, about 560 amino acids, about 540amino acids, about 520 amino acids, about 500 amino acids, or about 490amino acids (inclusive); about 490 amino acids to about 800 amino acids,about 780 amino acids, about 760 amino acids, about 740 amino acids,about 720 amino acids, about 700 amino acids, about 680 amino acids,about 660 amino acids, about 640 amino acids, about 620 amino acids,about 600 amino acids, about 580 amino acids, about 560 amino acids,about 540 amino acids, about 520 amino acids, or about 500 amino acids(inclusive); about 500 amino acids to about 800 amino acids, about 780amino acids, about 760 amino acids, about 740 amino acids, about 720amino acids, about 700 amino acids, about 680 amino acids, about 660amino acids, about 640 amino acids, about 620 amino acids, about 600amino acids, about 580 amino acids, about 560 amino acids, about 540amino acids, or about 520 amino acids (inclusive); about 520 amino acidsto about 800 amino acids, about 780 amino acids, about 760 amino acids,about 740 amino acids, about 720 amino acids, about 700 amino acids,about 680 amino acids, about 660 amino acids, about 640 amino acids,about 620 amino acids, about 600 amino acids, about 580 amino acids,about 560 amino acids, or about 540 amino acids (inclusive); about 540amino acids to about 800 amino acids, about 780 amino acids, about 760amino acids, about 740 amino acids, about 720 amino acids, about 700amino acids, about 680 amino acids, about 660 amino acids, about 640amino acids, about 620 amino acids, about 600 amino acids, about 580amino acids, or about 560 amino acids (inclusive); about 560 amino acidsto about 800 amino acids, about 780 amino acids, about 760 amino acids,about 740 amino acids, about 720 amino acids, about 700 amino acids,about 680 amino acids, about 660 amino acids, about 640 amino acids,about 620 amino acids, about 600 amino acids, or about 580 amino acids(inclusive); about 580 amino acids to about 800 amino acids, about 780amino acids, about 760 amino acids, about 740 amino acids, about 720amino acids, about 700 amino acids, about 680 amino acids, about 660amino acids, about 640 amino acids, about 620 amino acids, or about 600amino acids (inclusive); about 600 amino acids to about 800 amino acids,about 780 amino acids, about 760 amino acids, about 740 amino acids,about 720 amino acids, about 700 amino acids, about 680 amino acids,about 660 amino acids, about 640 amino acids, or about 620 amino acids(inclusive); about 620 amino acids to about 800 amino acids, about 780amino acids, about 760 amino acids, about 740 amino acids, about 720amino acids, about 700 amino acids, about 680 amino acids, about 660amino acids, or about 640 amino acids (inclusive); about 640 amino acidsto about 800 amino acids, about 780 amino acids, about 760 amino acids,about 740 amino acids, about 720 amino acids, about 700 amino acids,about 680 amino acids, or about 660 amino acids (inclusive); about 660amino acids to about 800 amino acids, about 780 amino acids, about 760amino acids, about 740 amino acids, about 720 amino acids, about 700amino acids, or about 680 amino acids (inclusive); about 680 amino acidsto about 800 amino acids, about 780 amino acids, about 760 amino acids,about 740 amino acids, about 720 amino acids, or about 700 amino acids(inclusive); about 700 amino acids to about 800 amino acids, about 780amino acids, about 760 amino acids, about 740 amino acids, or about 720amino acids (inclusive); about 720 amino acids to about 800 amino acids,about 780 amino acids, about 760 amino acids, or about 740 amino acids(inclusive); about 740 amino acids to about 800 amino acids, about 780amino acids, or about 760 amino acids (inclusive); about 760 amino acidsto about 800 amino acids, or about 780 amino acids; (inclusive); orabout 780 amino acids to about 800 amino acids (inclusive).

In some embodiments, one or more intervening amino acids between thetransmembrane domain and the hormone receptor ligand binding domain canbe or include a linker peptide. In some embodiments, a linker peptidecan include one or more of the following amino acid sequences:GSGSGSGSGS (SEQ ID NO: 20), GSGSGSGS (SEQ ID NO: 21), RSGSGSGS (SEQ IDNO: 22), or GSGSGSGS (SEQ ID NO: 23). In some embodiments, linkerpeptide can be encoded by one or more of the following nucleic acidsequences: GGATCCGGCAGCGGATCTGGCAGTGGAAGC (SEQ ID NO: 24),GGATCTGGCTCTGGAAGCGGCAGC (SEQ ID NO: 25), AGATCCGGATCTGGAAGTGGCTCC (SEQID NO: 26), or GGAAGTGGATCTGGGAGCGGCTCT (SEQ ID NO: 27). In someembodiments, a linker peptide can include one or more (e.g., two, three,or four) copies of any one of SEQ ID NOs: 20-23, e.g., in tandem.

In some embodiments, one or more intervening amino acids between thetransmembrane domain and the hormone receptor ligand binding domain canbe or include an intracellular sequence that abuts the transmembranedomain in the endogenous polypeptide from which the transmembrane domain(e.g., any of the transmembrane domains described herein) is derived. Insome embodiments, one or more intervening amino acids between thetransmembrane domain and the hormone receptor ligand binding domain canbe or include one or more additional domains (e.g., any of the exemplaryadditional domains described herein or known in the art). In someembodiments, a single-chain chimeric polypeptide can be a single-chainchimeric antigen receptor (e.g., any of the single-chain chimericantigen receptors described herein). In some embodiments, the chimericpolypeptide can further include one or more additional domains.Non-limiting examples of additional domains that can be included in asingle-chain chimeric polypeptide include one or more of: anextracellular ligand binding domain (e.g., an antigen-binding domain,e.g., any of the antigen-binding domains described herein), an ITIM(e.g., any of the ITIMs known in the art), an ITAM (e.g., any of ITAMsdescribed herein or known in the art), a dimerization domain (e.g.,capable of dimerizing with another dimerization domain present inanother polypeptide (e.g., a recombinant polypeptide) expressed in amammalian cell, e.g., any of the dimerization domains known in the art),and a peptide tag (e.g., a poly-His tag).

Single-Chain Chimeric Polypeptide—Extracellular Hormone Receptor LBD

Provided herein are single-chain chimeric polypeptides that include anextracellular hormone receptor ligand binding domain (e.g., any of thehormone receptor ligand binding domains described herein or known in theart) and a transmembrane domain (e.g., any of the transmembrane domainsdescribed herein or known in the art), where the transmembrane domainand the extracellular hormone receptor ligand binding domain directlyabut each other or are separated by 1 to about 800 amino acids (e.g., orany of the subranges of this range described herein), and thetransmembrane domain and the hormone receptor ligand binding domain arenot both present in a single endogenous single-chain polypeptide in amammal (e.g., a human).

In some examples of the single-chain chimeric polypeptides, thetransmembrane domain directly abuts the hormone receptor ligand bindingdomain (i.e., no intervening amino acids between the transmembranedomain and the extracellular hormone receptor ligand binding domain). Insome examples of the single-chain chimeric polypeptides, thetransmembrane domain and the extracellular hormone receptor ligandbinding domain are separated by: 1 amino acid to about 800 amino acids(e.g., any of the subranges of this range described herein).

In some embodiments, one or more intervening amino acids between thetransmembrane domain and the extracellular hormone receptor ligandbinding domain can be or include a linker peptide. In some embodiments,a linker peptide can include one or more of the following amino acidsequences: GSGSGSGSGS (SEQ ID NO: 20), GSGSGSGS (SEQ ID NO: 21),RSGSGSGS (SEQ ID NO: 22), or GSGSGSGS (SEQ ID NO: 23). In someembodiments, linker peptide can be encoded by one or more of thefollowing nucleic acid sequences: GGATCCGGCAGCGGATCTGGCAGTGGAAGC (SEQ IDNO: 24), GGATCTGGCTCTGGAAGCGGCAGC (SEQ ID NO: 25),AGATCCGGATCTGGAAGTGGCTCC (SEQ ID NO: 26), or GGAAGTGGATCTGGGAGCGGCTCT(SEQ ID NO: 27). In some embodiments, a linker peptide can include oneor more (e.g., two, three, or four) copies of any one of SEQ ID NOs:20-23, e.g., in tandem. In some embodiments, a linker peptide can be orcan include one or more of SEQ ID NO: 45, 47, 49, 58, 60, 62, and 106.In some embodiments, a linker peptide can be or include GS.

In some embodiments, one or more intervening amino acids between thetransmembrane domain and the extracellular hormone receptor ligandbinding domain can be or include a sequence that abuts the transmembranedomain in the endogenous polypeptide from which the transmembrane domain(e.g., any of the transmembrane domains described herein) is derived. Insome embodiments, one or more intervening amino acids between thetransmembrane domain and the extracellular hormone receptor ligandbinding domain can be or include one or more additional domains (e.g.,any of the exemplary additional domains described herein or known in theart). In some embodiments, a single-chain chimeric polypeptide can be asingle-chain chimeric antigen receptor (e.g., any of the single-chainchimeric antigen receptors described herein). In some embodiments, thechimeric polypeptide can further include one or more (e.g., two, three,four, or five) additional domains. Non-limiting examples of additionaldomains that can be included in a single-chain chimeric polypeptideinclude one or more of: an extracellular ligand binding domain (e.g., anantigen-binding domain, e.g., any of the antigen-binding domainsdescribed herein), an ITIM (e.g., any of the ITIMs known in the art), anITAM (e.g., any of ITAMs described herein or known in the art), adimerization domain (e.g., capable of dimerizing with anotherdimerization domain present in another polypeptide (e.g., a recombinantpolypeptide) expressed in a mammalian cell, e.g., any of thedimerization domains known in the art), and a peptide tag (e.g., apoly-His tag).

In some embodiments, when going in the N-terminal to C-terminaldirection, the single-chain chimeric polypeptide includes theextracellular antigen-binding domain, the extracellular hormone receptorligand binding domain, and the transmembrane domain. In someembodiments, when going in the C-terminal to the N-terminal direction,the single-chain chimeric polypeptide comprises the extracellularantigen-binding domain, the extracellular hormone receptor ligandbinding domain, and the transmembrane domain. In some embodiments, whengoing in the N-terminal to the C-terminal direction, the single-chainchimeric polypeptide includes the extracellular hormone receptor ligandbinding domain, the extracellular antigen-binding domain, and thetransmembrane domain. In some embodiments, when going in the C-terminalto the N-terminal direction, the single-chain chimeric polypeptideincludes the extracellular hormone receptor ligand binding domain, theextracellular antigen-binding domain, and the transmembrane domain.

Single-Chain Chimeric Antigen Receptor

Single-Chain Chimeric Antigen Receptor—Intracellular Hormone ReceptorLBD

Also provided herein are single-chain chimeric antigen receptors thatinclude: an extracellular antigen-binding domain (e.g., any of theantigen-binding domains described herein or known in the art), atransmembrane domain (e.g., any of the transmembrane domains describedherein or known in the art), a hormone receptor ligand binding domain(e.g., any of the hormone receptor ligand binding domain describedherein or known in the art), a costimulatory domain (e.g., any of thecostimulatory domains described herein or known in the art), and animmunoreceptor tyrosine-based activation motif (ITAM), where: thetransmembrane domain and the hormone ligand binding domain directly abuteach other or are separated by 1 to about 800 amino acids (e.g., any ofthe subranges of this range described herein), and the transmembranedomain and the hormone receptor ligand binding domain are not bothpresent in the same (a single) endogenous single-chain polypeptide in amammal. The single-chain chimeric antigen receptors described herein canbind to any of the exemplary antigens described herein or any otherantigen known in the art. In some embodiments, the single-chain chimericantigen receptor binds specifically to a single antigen (e.g., any ofthe exemplary antigens described herein). In some embodiments, thesingle-chain chimeric antigen receptor binds specifically to twodifferent antigens (e.g., any combination of the exemplary antigensdescribed herein).

In some embodiments of these single-chain chimeric antigen receptors,the transmembrane domain abuts the hormone receptor ligand bindingdomain. In some embodiments of these single-chain chimeric antigenreceptors, the transmembrane domain and the hormone receptor ligandbinding domain are separated by 1 to about 800 amino acids (e.g., or anyof the subranges of this range described herein). In some embodiments,the amino acids between the transmembrane domain and the hormonereceptor ligand binding domain do not comprise a domain. In otherembodiments, the amino acids between the transmembrane domain and thehormone receptor ligand binding domain includes one or more interveningdomain (e.g., one or more costimulatory domains and/or one or moreITAMs). In some embodiments, the amino acids between the transmembranedomain and the hormone receptor ligand binding domain includes asequence from the same endogenous transmembrane protein from which thetransmembrane domain is derived. In some embodiments, the amino acidsbetween the transmembrane domain and the hormone receptor ligand bindingdomain include a linker sequence.

Some embodiments of these single-chain chimeric antigen receptors caninclude one or more (e.g., two, three, four, or five) costimulatorydomain(s) (e.g., any combination of any of the exemplary costimulatorydomains described herein or known in the art). Some embodiments of thesesingle-chain chimeric antigen receptors include one or both of a 4-1BBcostimulatory domain and a CD28 costimulatory domain.

Some embodiments of these single-chain chimeric antigen receptors caninclude one or more (e.g., two, three, four, or five) ITAMs (e.g., anyof the ITAMs described herein or known in the art). In some embodimentsof these single-chain chimeric antigen receptors, the ITAM includes acytoplasmic signaling sequence from CD3ζ (e.g., human CD3ζ).

In some embodiments of any of these single-chain chimeric antigenreceptors, any two neighboring domains can be separated by 1 amino acidsto about 500 amino acids, about 480 amino acids, about 460 amino acids,about 440 amino acids, about 420 amino acids, about 400 amino acids,about 380 amino acids, about 360 amino acids, about 340 amino acids,about 320 amino acids, about 300 amino acids, about 280 amino acids,about 260 amino acids, about 240 amino acids, about 220 amino acids,about 200 amino acids, about 190 amino acids, about 180 amino acids,about 170 amino acids, about 160 amino acids, about 150 amino acids,about 140 amino acids, about 130 amino acids, about 120 amino acids,about 110 amino acids, about 100 amino acids, about 95 amino acids,about 90 amino acids, about 85 amino acids, about 80 amino acids, about75 amino acids, about 70 amino acids, about 65 amino acids, about 60amino acids, about 55 amino acids, about 50 amino acids, about 45 aminoacids, about 40 amino acids, about 35 amino acids, about 30 amino acids,about 25 amino acids, about 20 amino acids, about 15 amino acids, about10 amino acids, about 8 amino acids, about 6 amino acids, about 4 aminoacid, or about 3 amino acids (inclusive); about 2 amino acids to about500 amino acids, about 480 amino acids, about 460 amino acids, about 440amino acids, about 420 amino acids, about 400 amino acids, about 380amino acids, about 360 amino acids, about 340 amino acids, about 320amino acids, about 300 amino acids, about 280 amino acids, about 260amino acids, about 240 amino acids, about 220 amino acids, about 200amino acids, about 190 amino acids, about 180 amino acids, about 170amino acids, about 160 amino acids, about 150 amino acids, about 140amino acids, about 130 amino acids, about 120 amino acids, about 110amino acids, about 100 amino acids, about 95 amino acids, about 90 aminoacids, about 85 amino acids, about 80 amino acids, about 75 amino acids,about 70 amino acids, about 65 amino acids, about 60 amino acids, about55 amino acids, about 50 amino acids, about 45 amino acids, about 40amino acids, about 35 amino acids, about 30 amino acids, about 25 aminoacids, about 20 amino acids, about 15 amino acids, about 10 amino acids,about 8 amino acids, about 6 amino acids, or about 4 amino acid(inclusive); about 3 amino acids to about 500 amino acids, about 480amino acids, about 460 amino acids, about 440 amino acids, about 420amino acids, about 400 amino acids, about 380 amino acids, about 360amino acids, about 340 amino acids, about 320 amino acids, about 300amino acids, about 280 amino acids, about 260 amino acids, about 240amino acids, about 220 amino acids, about 200 amino acids, about 190amino acids, about 180 amino acids, about 170 amino acids, about 160amino acids, about 150 amino acids, about 140 amino acids, about 130amino acids, about 120 amino acids, about 110 amino acids, about 100amino acids, about 95 amino acids, about 90 amino acids, about 85 aminoacids, about 80 amino acids, about 75 amino acids, about 70 amino acids,about 65 amino acids, about 60 amino acids, about 55 amino acids, about50 amino acids, about 45 amino acids, about 40 amino acids, about 35amino acids, about 30 amino acids, about 25 amino acids, about 20 aminoacids, about 15 amino acids, about 10 amino acids, about 8 amino acids,about 6 amino acids, or about 5 amino acids (inclusive); about 4 aminoacids to about 500 amino acids, about 480 amino acids, about 460 aminoacids, about 440 amino acids, about 420 amino acids, about 400 aminoacids, about 380 amino acids, about 360 amino acids, about 340 aminoacids, about 320 amino acids, about 300 amino acids, about 280 aminoacids, about 260 amino acids, about 240 amino acids, about 220 aminoacids, about 200 amino acids, about 190 amino acids, about 180 aminoacids, about 170 amino acids, about 160 amino acids, about 150 aminoacids, about 140 amino acids, about 130 amino acids, about 120 aminoacids, about 110 amino acids, about 100 amino acids, about 95 aminoacids, about 90 amino acids, about 85 amino acids, about 80 amino acids,about 75 amino acids, about 70 amino acids, about 65 amino acids, about60 amino acids, about 55 amino acids, about 50 amino acids, about 45amino acids, about 40 amino acids, about 35 amino acids, about 30 aminoacids, about 25 amino acids, about 20 amino acids, about 15 amino acids,about 10 amino acids, about 8 amino acids, or about 6 amino acids(inclusive); about 5 amino acids to about 500 amino acids, about 480amino acids, about 460 amino acids, about 440 amino acids, about 420amino acids, about 400 amino acids, about 380 amino acids, about 360amino acids, about 340 amino acids, about 320 amino acids, about 300amino acids, about 280 amino acids, about 260 amino acids, about 240amino acids, about 220 amino acids, about 200 amino acids, about 190amino acids, about 180 amino acids, about 170 amino acids, about 160amino acids, about 150 amino acids, about 140 amino acids, about 130amino acids, about 120 amino acids, about 110 amino acids, about 100amino acids, about 95 amino acids, about 90 amino acids, about 85 aminoacids, about 80 amino acids, about 75 amino acids, about 70 amino acids,about 65 amino acids, about 60 amino acids, about 55 amino acids, about50 amino acids, about 45 amino acids, about 40 amino acids, about 35amino acids, about 30 amino acids, about 25 amino acids, about 20 aminoacids, about 15 amino acids, about 10 amino acids, about 8 amino acids,or about 7 amino acids (inclusive); about 6 amino acids to about 500amino acids, about 480 amino acids, about 460 amino acids, about 440amino acids, about 420 amino acids, about 400 amino acids, about 380amino acids, about 360 amino acids, about 340 amino acids, about 320amino acids, about 300 amino acids, about 280 amino acids, about 260amino acids, about 240 amino acids, about 220 amino acids, about 200amino acids, about 190 amino acids, about 180 amino acids, about 170amino acids, about 160 amino acids, about 150 amino acids, about 140amino acids, about 130 amino acids, about 120 amino acids, about 110amino acids, about 100 amino acids, about 95 amino acids, about 90 aminoacids, about 85 amino acids, about 80 amino acids, about 75 amino acids,about 70 amino acids, about 65 amino acids, about 60 amino acids, about55 amino acids, about 50 amino acids, about 45 amino acids, about 40amino acids, about 35 amino acids, about 30 amino acids, about 25 aminoacids, about 20 amino acids, about 15 amino acids, about 10 amino acids,or about 8 amino acids (inclusive); about 8 amino acids to about 500amino acids, about 480 amino acids, about 460 amino acids, about 440amino acids, about 420 amino acids, about 400 amino acids, about 380amino acids, about 360 amino acids, about 340 amino acids, about 320amino acids, about 300 amino acids, about 280 amino acids, about 260amino acids, about 240 amino acids, about 220 amino acids, about 200amino acids, about 190 amino acids, about 180 amino acids, about 170amino acids, about 160 amino acids, about 150 amino acids, about 140amino acids, about 130 amino acids, about 120 amino acids, about 110amino acids, about 100 amino acids, about 95 amino acids, about 90 aminoacids, about 85 amino acids, about 80 amino acids, about 75 amino acids,about 70 amino acids, about 65 amino acids, about 60 amino acids, about55 amino acids, about 50 amino acids, about 45 amino acids, about 40amino acids, about 35 amino acids, about 30 amino acids, about 25 aminoacids, about 20 amino acids, about 15 amino acids, or about 10 aminoacids (inclusive); about 10 amino acids to about 500 amino acids, about480 amino acids, about 460 amino acids, about 440 amino acids, about 420amino acids, about 400 amino acids, about 380 amino acids, about 360amino acids, about 340 amino acids, about 320 amino acids, about 300amino acids, about 280 amino acids, about 260 amino acids, about 240amino acids, about 220 amino acids, about 200 amino acids, about 190amino acids, about 180 amino acids, about 170 amino acids, about 160amino acids, about 150 amino acids, about 140 amino acids, about 130amino acids, about 120 amino acids, about 110 amino acids, about 100amino acids, about 95 amino acids, about 90 amino acids, about 85 aminoacids, about 80 amino acids, about 75 amino acids, about 70 amino acids,about 65 amino acids, about 60 amino acids, about 55 amino acids, about50 amino acids, about 45 amino acids, about 40 amino acids, about 35amino acids, about 30 amino acids, about 25 amino acids, about 20 aminoacids, or about 15 amino acids (inclusive); about 15 amino acids toabout 500 amino acids, about 480 amino acids, about 460 amino acids,about 440 amino acids, about 420 amino acids, about 400 amino acids,about 380 amino acids, about 360 amino acids, about 340 amino acids,about 320 amino acids, about 300 amino acids, about 280 amino acids,about 260 amino acids, about 240 amino acids, about 220 amino acids,about 200 amino acids, about 190 amino acids, about 180 amino acids,about 170 amino acids, about 160 amino acids, about 150 amino acids,about 140 amino acids, about 130 amino acids, about 120 amino acids,about 110 amino acids, about 100 amino acids, about 95 amino acids,about 90 amino acids, about 85 amino acids, about 80 amino acids, about75 amino acids, about 70 amino acids, about 65 amino acids, about 60amino acids, about 55 amino acids, about 50 amino acids, about 45 aminoacids, about 40 amino acids, about 35 amino acids, about 30 amino acids,about 25 amino acids, or about 20 amino acids (inclusive); about 20amino acids to about 500 amino acids, about 480 amino acids, about 460amino acids, about 440 amino acids, about 420 amino acids, about 400amino acids, about 380 amino acids, about 360 amino acids, about 340amino acids, about 320 amino acids, about 300 amino acids, about 280amino acids, about 260 amino acids, about 240 amino acids, about 220amino acids, about 200 amino acids, about 190 amino acids, about 180amino acids, about 170 amino acids, about 160 amino acids, about 150amino acids, about 140 amino acids, about 130 amino acids, about 120amino acids, about 110 amino acids, about 100 amino acids, about 95amino acids, about 90 amino acids, about 85 amino acids, about 80 aminoacids, about 75 amino acids, about 70 amino acids, about 65 amino acids,about 60 amino acids, about 55 amino acids, about 50 amino acids, about45 amino acids, about 40 amino acids, about 35 amino acids, about 30amino acids, or about 25 amino acids (inclusive); about 25 amino acidsto about 500 amino acids, about 480 amino acids, about 460 amino acids,about 440 amino acids, about 420 amino acids, about 400 amino acids,about 380 amino acids, about 360 amino acids, about 340 amino acids,about 320 amino acids, about 300 amino acids, about 280 amino acids,about 260 amino acids, about 240 amino acids, about 220 amino acids,about 200 amino acids, about 190 amino acids, about 180 amino acids,about 170 amino acids, about 160 amino acids, about 150 amino acids,about 140 amino acids, about 130 amino acids, about 120 amino acids,about 110 amino acids, about 100 amino acids, about 95 amino acids,about 90 amino acids, about 85 amino acids, about 80 amino acids, about75 amino acids, about 70 amino acids, about 65 amino acids, about 60amino acids, about 55 amino acids, about 50 amino acids, about 45 aminoacids, about 40 amino acids, about 35 amino acids, or about 30 aminoacids (inclusive); about 30 amino acids to about 500 amino acids, about480 amino acids, about 460 amino acids, about 440 amino acids, about 420amino acids, about 400 amino acids, about 380 amino acids, about 360amino acids, about 340 amino acids, about 320 amino acids, about 300amino acids, about 280 amino acids, about 260 amino acids, about 240amino acids, about 220 amino acids, about 200 amino acids, about 190amino acids, about 180 amino acids, about 170 amino acids, about 160amino acids, about 150 amino acids, about 140 amino acids, about 130amino acids, about 120 amino acids, about 110 amino acids, about 100amino acids, about 95 amino acids, about 90 amino acids, about 85 aminoacids, about 80 amino acids, about 75 amino acids, about 70 amino acids,about 65 amino acids, about 60 amino acids, about 55 amino acids, about50 amino acids, about 45 amino acids, about 40 amino acids, or about 35amino acids (inclusive); about 35 amino acids to about 500 amino acids,about 480 amino acids, about 460 amino acids, about 440 amino acids,about 420 amino acids, about 400 amino acids, about 380 amino acids,about 360 amino acids, about 340 amino acids, about 320 amino acids,about 300 amino acids, about 280 amino acids, about 260 amino acids,about 240 amino acids, about 220 amino acids, about 200 amino acids,about 190 amino acids, about 180 amino acids, about 170 amino acids,about 160 amino acids, about 150 amino acids, about 140 amino acids,about 130 amino acids, about 120 amino acids, about 110 amino acids,about 100 amino acids, about 95 amino acids, about 90 amino acids, about85 amino acids, about 80 amino acids, about 75 amino acids, about 70amino acids, about 65 amino acids, about 60 amino acids, about 55 aminoacids, about 50 amino acids, about 45 amino acids, about 40 amino acids(inclusive); about 40 amino acids to about 500 amino acids, about 480amino acids, about 460 amino acids, about 440 amino acids, about 420amino acids, about 400 amino acids, about 380 amino acids, about 360amino acids, about 340 amino acids, about 320 amino acids, about 300amino acids, about 280 amino acids, about 260 amino acids, about 240amino acids, about 220 amino acids, about 200 amino acids, about 190amino acids, about 180 amino acids, about 170 amino acids, about 160amino acids, about 150 amino acids, about 140 amino acids, about 130amino acids, about 120 amino acids, about 110 amino acids, about 100amino acids, about 95 amino acids, about 90 amino acids, about 85 aminoacids, about 80 amino acids, about 75 amino acids, about 70 amino acids,about 65 amino acids, about 60 amino acids, about 55 amino acids, about50 amino acids, or about 45 amino acids (inclusive); about 45 aminoacids to about 500 amino acids, about 480 amino acids, about 460 aminoacids, about 440 amino acids, about 420 amino acids, about 400 aminoacids, about 380 amino acids, about 360 amino acids, about 340 aminoacids, about 320 amino acids, about 300 amino acids, about 280 aminoacids, about 260 amino acids, about 240 amino acids, about 220 aminoacids, about 200 amino acids, about 190 amino acids, about 180 aminoacids, about 170 amino acids, about 160 amino acids, about 150 aminoacids, about 140 amino acids, about 130 amino acids, about 120 aminoacids, about 110 amino acids, about 100 amino acids, about 95 aminoacids, about 90 amino acids, about 85 amino acids, about 80 amino acids,about 75 amino acids, about 70 amino acids, about 65 amino acids, about60 amino acids, about 55 amino acids, or about 50 amino acids(inclusive); about 50 amino acids to about 500 amino acids, about 480amino acids, about 460 amino acids, about 440 amino acids, about 420amino acids, about 400 amino acids, about 380 amino acids, about 360amino acids, about 340 amino acids, about 320 amino acids, about 300amino acids, about 280 amino acids, about 260 amino acids, about 240amino acids, about 220 amino acids, about 200 amino acids, about 190amino acids, about 180 amino acids, about 170 amino acids, about 160amino acids, about 150 amino acids, about 140 amino acids, about 130amino acids, about 120 amino acids, about 110 amino acids, about 100amino acids, about 95 amino acids, about 90 amino acids, about 85 aminoacids, about 80 amino acids, about 75 amino acids, about 70 amino acids,about 65 amino acids, about 60 amino acids, or about 55 amino acids(inclusive); about 55 amino acids to about 500 amino acids, about 480amino acids, about 460 amino acids, about 440 amino acids, about 420amino acids, about 400 amino acids, about 380 amino acids, about 360amino acids, about 340 amino acids, about 320 amino acids, about 300amino acids, about 280 amino acids, about 260 amino acids, about 240amino acids, about 220 amino acids, about 200 amino acids, about 190amino acids, about 180 amino acids, about 170 amino acids, about 160amino acids, about 150 amino acids, about 140 amino acids, about 130amino acids, about 120 amino acids, about 110 amino acids, about 100amino acids, about 95 amino acids, about 90 amino acids, about 85 aminoacids, about 80 amino acids, about 75 amino acids, about 70 amino acids,about 65 amino acids, or about 60 amino acids (inclusive); about 60amino acids to about 500 amino acids, about 480 amino acids, about 460amino acids, about 440 amino acids, about 420 amino acids, about 400amino acids, about 380 amino acids, about 360 amino acids, about 340amino acids, about 320 amino acids, about 300 amino acids, about 280amino acids, about 260 amino acids, about 240 amino acids, about 220amino acids, about 200 amino acids, about 190 amino acids, about 180amino acids, about 170 amino acids, about 160 amino acids, about 150amino acids, about 140 amino acids, about 130 amino acids, about 120amino acids, about 110 amino acids, about 100 amino acids, about 95amino acids, about 90 amino acids, about 85 amino acids, about 80 aminoacids, about 75 amino acids, about 70 amino acids, or about 65 aminoacids (inclusive); about 65 amino acids to about 500 amino acids, about480 amino acids, about 460 amino acids, about 440 amino acids, about 420amino acids, about 400 amino acids, about 380 amino acids, about 360amino acids, about 340 amino acids, about 320 amino acids, about 300amino acids, about 280 amino acids, about 260 amino acids, about 240amino acids, about 220 amino acids, about 200 amino acids, about 190amino acids, about 180 amino acids, about 170 amino acids, about 160amino acids, about 150 amino acids, about 140 amino acids, about 130amino acids, about 120 amino acids, about 110 amino acids, about 100amino acids, about 95 amino acids, about 90 amino acids, about 85 aminoacids, about 80 amino acids, about 75 amino acids, or about 70 aminoacids (inclusive); about 70 amino acids to about 500 amino acids, about480 amino acids, about 460 amino acids, about 440 amino acids, about 420amino acids, about 400 amino acids, about 380 amino acids, about 360amino acids, about 340 amino acids, about 320 amino acids, about 300amino acids, about 280 amino acids, about 260 amino acids, about 240amino acids, about 220 amino acids, about 200 amino acids, about 190amino acids, about 180 amino acids, about 170 amino acids, about 160amino acids, about 150 amino acids, about 140 amino acids, about 130amino acids, about 120 amino acids, about 110 amino acids, about 100amino acids, about 95 amino acids, about 90 amino acids, about 85 aminoacids, about 80 amino acids, or about 75 amino acids (inclusive); about75 amino acids to about 500 amino acids, about 480 amino acids, about460 amino acids, about 440 amino acids, about 420 amino acids, about 400amino acids, about 380 amino acids, about 360 amino acids, about 340amino acids, about 320 amino acids, about 300 amino acids, about 280amino acids, about 260 amino acids, about 240 amino acids, about 220amino acids, about 200 amino acids, about 190 amino acids, about 180amino acids, about 170 amino acids, about 160 amino acids, about 150amino acids, about 140 amino acids, about 130 amino acids, about 120amino acids, about 110 amino acids, about 100 amino acids, about 95amino acids, about 90 amino acids, about 85 amino acids, about 80 aminoacids, or about 75 amino acids (inclusive); about 75 amino acids toabout 500 amino acids, about 480 amino acids, about 460 amino acids,about 440 amino acids, about 420 amino acids, about 400 amino acids,about 380 amino acids, about 360 amino acids, about 340 amino acids,about 320 amino acids, about 300 amino acids, about 280 amino acids,about 260 amino acids, about 240 amino acids, about 220 amino acids,about 200 amino acids, about 190 amino acids, about 180 amino acids,about 170 amino acids, about 160 amino acids, about 150 amino acids,about 140 amino acids, about 130 amino acids, about 120 amino acids,about 110 amino acids, about 100 amino acids, about 95 amino acids,about 90 amino acids, about 85 amino acids, or about 80 amino acids(inclusive); about 80 amino acids to about 500 amino acids, about 480amino acids, about 460 amino acids, about 440 amino acids, about 420amino acids, about 400 amino acids, about 380 amino acids, about 360amino acids, about 340 amino acids, about 320 amino acids, about 300amino acids, about 280 amino acids, about 260 amino acids, about 240amino acids, about 220 amino acids, about 200 amino acids, about 190amino acids, about 180 amino acids, about 170 amino acids, about 160amino acids, about 150 amino acids, about 140 amino acids, about 130amino acids, about 120 amino acids, about 110 amino acids, about 100amino acids, about 95 amino acids, or about 90 amino acids (inclusive);about 100 amino acids to about 500 amino acids, about 480 amino acids,about 460 amino acids, about 440 amino acids, about 420 amino acids,about 400 amino acids, about 380 amino acids, about 360 amino acids,about 340 amino acids, about 320 amino acids, about 300 amino acids,about 280 amino acids, about 260 amino acids, about 240 amino acids,about 220 amino acids, about 200 amino acids, about 190 amino acids,about 180 amino acids, about 170 amino acids, about 160 amino acids,about 150 amino acids, about 140 amino acids, about 130 amino acids,about 120 amino acids, or about 110 amino acids (inclusive); about 110amino acids to about 500 amino acids, about 480 amino acids, about 460amino acids, about 440 amino acids, about 420 amino acids, about 400amino acids, about 380 amino acids, about 360 amino acids, about 340amino acids, about 320 amino acids, about 300 amino acids, about 280amino acids, about 260 amino acids, about 240 amino acids, about 220amino acids, about 200 amino acids, about 190 amino acids, about 180amino acids, about 170 amino acids, about 160 amino acids, about 150amino acids, about 140 amino acids, about 130 amino acids, or about 120amino acids (inclusive); about 120 amino acids to about 500 amino acids,about 480 amino acids, about 460 amino acids, about 440 amino acids,about 420 amino acids, about 400 amino acids, about 380 amino acids,about 360 amino acids, about 340 amino acids, about 320 amino acids,about 300 amino acids, about 280 amino acids, about 260 amino acids,about 240 amino acids, about 220 amino acids, about 200 amino acids,about 190 amino acids, about 180 amino acids, about 170 amino acids,about 160 amino acids, about 150 amino acids, about 140 amino acids,about 130 amino acids, or about 120 amino acids (inclusive); about 120amino acids to about 500 amino acids, about 480 amino acids, about 460amino acids, about 440 amino acids, about 420 amino acids, about 400amino acids, about 380 amino acids, about 360 amino acids, about 340amino acids, about 320 amino acids, about 300 amino acids, about 280amino acids, about 260 amino acids, about 240 amino acids, about 220amino acids, about 200 amino acids, about 190 amino acids, about 180amino acids, about 170 amino acids, about 160 amino acids, about 150amino acids, about 140 amino acids, or about 130 amino acids(inclusive); about 130 amino acids to about 500 amino acids, about 480amino acids, about 460 amino acids, about 440 amino acids, about 420amino acids, about 400 amino acids, about 380 amino acids, about 360amino acids, about 340 amino acids, about 320 amino acids, about 300amino acids, about 280 amino acids, about 260 amino acids, about 240amino acids, about 220 amino acids, about 200 amino acids, about 190amino acids, about 180 amino acids, about 170 amino acids, about 160amino acids, about 150 amino acids, or about 140 amino acids(inclusive); about 140 amino acids to about 500 amino acids, about 480amino acids, about 460 amino acids, about 440 amino acids, about 420amino acids, about 400 amino acids, about 380 amino acids, about 360amino acids, about 340 amino acids, about 320 amino acids, about 300amino acids, about 280 amino acids, about 260 amino acids, about 240amino acids, about 220 amino acids, about 200 amino acids, about 190amino acids, about 180 amino acids, about 170 amino acids, about 160amino acids, or about 150 amino acids (inclusive); about 150 amino acidsto about 500 amino acids, about 480 amino acids, about 460 amino acids,about 440 amino acids, about 420 amino acids, about 400 amino acids,about 380 amino acids, about 360 amino acids, about 340 amino acids,about 320 amino acids, about 300 amino acids, about 280 amino acids,about 260 amino acids, about 240 amino acids, about 220 amino acids,about 200 amino acids, about 190 amino acids, about 180 amino acids,about 170 amino acids, or about 160 amino acids (inclusive); about 160amino acids to about 500 amino acids, about 480 amino acids, about 460amino acids, about 440 amino acids, about 420 amino acids, about 400amino acids, about 380 amino acids, about 360 amino acids, about 340amino acids, about 320 amino acids, about 300 amino acids, about 280amino acids, about 260 amino acids, about 240 amino acids, about 220amino acids, about 200 amino acids, about 190 amino acids, about 180amino acids, or about 170 amino acids (inclusive); about 170 amino acidsto about 500 amino acids, about 480 amino acids, about 460 amino acids,about 440 amino acids, about 420 amino acids, about 400 amino acids,about 380 amino acids, about 360 amino acids, about 340 amino acids,about 320 amino acids, about 300 amino acids, about 280 amino acids,about 260 amino acids, about 240 amino acids, about 220 amino acids,about 200 amino acids, about 190 amino acids, or about 180 amino acids(inclusive); about 180 amino acids to about 500 amino acids, about 480amino acids, about 460 amino acids, about 440 amino acids, about 420amino acids, about 400 amino acids, about 380 amino acids, about 360amino acids, about 340 amino acids, about 320 amino acids, about 300amino acids, about 280 amino acids, about 260 amino acids, about 240amino acids, about 220 amino acids, about 200 amino acids, or about 190amino acids (inclusive); about 190 amino acids to about 500 amino acids,about 480 amino acids, about 460 amino acids, about 440 amino acids,about 420 amino acids, about 400 amino acids, about 380 amino acids,about 360 amino acids, about 340 amino acids, about 320 amino acids,about 300 amino acids, about 280 amino acids, about 260 amino acids,about 240 amino acids, about 220 amino acids, or about 200 amino acids(inclusive); about 200 amino acids to about 500 amino acids, about 480amino acids, about 460 amino acids, about 440 amino acids, about 420amino acids, about 400 amino acids, about 380 amino acids, about 360amino acids, about 340 amino acids, about 320 amino acids, about 300amino acids, about 280 amino acids, about 260 amino acids, about 240amino acids, or about 220 amino acids (inclusive); about 220 amino acidsto about 500 amino acids, about 480 amino acids, about 460 amino acids,about 440 amino acids, about 420 amino acids, about 400 amino acids,about 380 amino acids, about 360 amino acids, about 340 amino acids,about 320 amino acids, about 300 amino acids, about 280 amino acids,about 260 amino acids, or about 240 amino acids (inclusive); about 240amino acids to about 500 amino acids, about 480 amino acids, about 460amino acids, about 440 amino acids, about 420 amino acids, about 400amino acids, about 380 amino acids, about 360 amino acids, about 340amino acids, about 320 amino acids, about 300 amino acids, about 280amino acids, or about 260 amino acids (inclusive); about 260 amino acidsto about 500 amino acids, about 480 amino acids, about 460 amino acids,about 440 amino acids, about 420 amino acids, about 400 amino acids,about 380 amino acids, about 360 amino acids, about 340 amino acids,about 320 amino acids, about 300 amino acids, or about 280 amino acids(inclusive); about 280 amino acids to about 500 amino acids, about 480amino acids, about 460 amino acids, about 440 amino acids, about 420amino acids, about 400 amino acids, about 380 amino acids, about 360amino acids, about 340 amino acids, about 320 amino acids, or about 300amino acids (inclusive); about 300 amino acids to about 500 amino acids,about 480 amino acids, about 460 amino acids, about 440 amino acids,about 420 amino acids, about 400 amino acids, about 380 amino acids,about 360 amino acids, about 340 amino acids, or about 320 amino acids(inclusive); about 320 amino acids to about 500 amino acids, about 480amino acids, about 460 amino acids, about 440 amino acids, about 420amino acids, about 400 amino acids, about 380 amino acids, about 360amino acids, or about 340 amino acids (inclusive); about 340 amino acidsto about 500 amino acids, about 480 amino acids, about 460 amino acids,about 440 amino acids, about 420 amino acids, about 400 amino acids,about 380 amino acids, or about 360 amino acids (inclusive); about 360amino acids to about 500 amino acids, about 480 amino acids, about 460amino acids, about 440 amino acids, about 420 amino acids, about 400amino acids, or about 380 amino acids (inclusive); about 380 amino acidsto about 500 amino acids, about 480 amino acids, about 460 amino acids,about 440 amino acids, about 420 amino acids, or about 400 amino acids(inclusive); about 400 amino acids to about 500 amino acids, about 480amino acids, about 460 amino acids, about 440 amino acids, or about 420amino acids (inclusive); about 420 amino acids to about 500 amino acids,about 480 amino acids, about 460 amino acids, or about 440 amino acids(inclusive); about 440 amino acids to about 500 amino acids, about 480amino acids, or about 460 amino acids (inclusive); about 460 amino acidsto about 500 amino acids or about 480 amino acids (inclusive); or about480 amino acids to about 500 amino acids (inclusive).

In some embodiments, one or more amino acids between the extracellularantigen-binding domain and the transmembrane domain is a sequence fromthe same endogenous single-chain polypeptide from which thetransmembrane domain is derived (e.g., a CD8a hinge region, e.g., SEQ IDNO: 112). In some embodiments, a sequence comprising SEQ ID NO: 42, 55,or 112 is positioned between the extracellular antigen-binding domainand the transmembrane domain. In some embodiments, one or more aminoacids between the extracellular antigen-binding domain and thetransmembrane domain is or includes a hinge region sequence of anantibody such as, without limitation, a human antibody (e.g., IgG1,IgG2, IgG3, or IgG4). In some embodiments, one or more amino acidsbetween the extracellular antigen-binding domain and the transmembranedomain is or comprises a linker sequence (e.g., a non-naturallyoccurring linker sequence, e.g., GS or any of the other linker sequencesdescribed herein).

In some embodiments of the single-chain chimeric antigen receptors,after the hormone receptor ligand binding domain, the next domain is acostimulatory domain followed by an ITAM. In some of these embodiments,one or more amino acids between the hormone receptor ligand bindingdomain and the costimulatory domain is or includes a sequence from thesame endogenous single-chain polypeptide from which the costimulatorydomain is derived or the ITAM is derived. In some of these embodiments,one or more amino acids between the hormone receptor ligand bindingdomain and the costimulatory domain is or includes a linker sequence(e.g., a non-naturally occurring linker sequence). In some embodiments,one or more amino acids between the costimulatory domain and the ITAM isor includes a sequence from the same endogenous single-chain polypeptidefrom which the costimulatory domain is derived or the ITAM is derived.

In some embodiments of the single-chain chimeric antigen receptors,after the hormone receptor ligand binding domain, the next domain is acostimulatory domain followed by an ITAM. In some embodiments of thesingle-chain chimeric antigen receptors, after the ligand bindingdomain, the next domain is an ITAM, followed by the costimulatorydomain. In some of these embodiments, one or more amino acids betweenthe hormone receptor ligand binding domain and the ITAM is or includes asequence from the same endogenous single-chain polypeptide from whichthe costimulatory domain is derived or the ITAM is derived. In some ofthese embodiments, one or more amino acids between the hormone receptorligand binding domain and the ITAM is or includes a linker sequence(e.g., a non-naturally occurring linker sequence). In some embodiments,one or more amino acids between the ITAM and the costimulatory domain isor includes a sequence from the same endogenous single-chain polypeptidefrom which the ITAM is derived or the costimulatory domain is derived.

In some embodiments, where two or more costimulatory domains areincluded in the single-chain chimeric antigen receptor, the two or morecostimulatory domains can be placed between the hormone receptor ligandbinding domain and one or more ITAMs. In some embodiments of thesingle-chain chimeric antigen receptors described herein, thecostimulatory domains and ITAMs can alternate in the primary amino acidsequence of the single-chain chimeric antigen receptor.

Some embodiments of any of the single-chain chimeric antigen receptorsdescribed herein can further include a dimerization domain and/or apeptide tag.

In some examples, a single-chain chimeric antigen receptor can comprisea sequence that is at least 80% (e.g., at least 82%, at least 84%, atleast 86%, at least 88%, at least 90%, at least 92%, at least 94%, atleast 96%, at least 98%, at least 99%, or 100%) identical to: SEQ ID NO:31, 33, 35, 37, 39, or 52.

Single-Chain Chimeric Antigen Receptor—Extracellular Hormone ReceptorLBD

Also provided herein are single-chain chimeric antigen receptors thatinclude: an extracellular antigen-binding domain (e.g., any of theantigen-binding domains described herein or known in the art), anextracellular hormone receptor ligand binding domain (e.g., any of thehormone receptor ligand binding domain described herein or known in theart), a transmembrane domain (e.g., any of the transmembrane domainsdescribed herein or known in the art), a costimulatory domain (e.g., anyof the costimulatory domains described herein or known in the art), andan immunoreceptor tyrosine-based activation motif (ITAM), where: thetransmembrane domain and the extracellular hormone ligand binding domaindirectly abut each other or are separated by 1 to about 800 amino acids(e.g., any of the subranges of this range described herein), and thetransmembrane domain and the extracellular hormone receptor ligandbinding domain are not present in the same endogenous single-chainpolypeptide in a mammal. The single-chain chimeric antigen receptorsdescribed herein can bind to any of the exemplary antigens describedherein or any other antigen known in the art. In some embodiments, thesingle-chain chimeric antigen receptor binds specifically to a singleantigen (e.g., any of the exemplary antigens described herein). In someembodiments, the single-chain chimeric antigen receptor bindsspecifically to two different antigens (e.g., any combination of theexemplary antigens described herein).

In some embodiments of these single-chain chimeric antigen receptors,the transmembrane domain abuts the extracellular hormone receptor ligandbinding domain. In some embodiments of these single-chain chimericantigen receptors, the transmembrane domain and the extracellularhormone receptor ligand binding domain are separated by 1 to about 800amino acids (e.g., or any of the subranges of this range describedherein). In some embodiments, the amino acids between the transmembranedomain and the extracellular hormone receptor ligand binding domain donot comprise a domain. In other embodiments, the amino acids between thetransmembrane domain and the extracellular hormone receptor ligandbinding domain includes one or more intervening domain (e.g., theantigen-binding domain or an additional antigen-binding domain). In someembodiments, the amino acids between the transmembrane domain and theextracellular hormone receptor ligand binding domain includes a sequencefrom the same endogenous transmembrane protein from which thetransmembrane domain is derived. In some embodiments, the amino acidsbetween the transmembrane domain and the extracellular hormone receptorligand binding domain include a linker sequence.

Some embodiments of these single-chain chimeric antigen receptors caninclude one or more (e.g., two, three, four, or five) costimulatorydomain(s) (e.g., any combination of any of the exemplary costimulatorydomains described herein or known in the art). Some embodiments of thesesingle-chain chimeric antigen receptors include one or both of a 4-1BBcostimulatory domain and a CD28 costimulatory domain.

Some embodiments of these single-chain chimeric antigen receptors caninclude one or more (e.g., two, three, four, or five) ITAMs (e.g., anyof the ITAMs described herein or known in the art). In some embodimentsof these single-chain chimeric antigen receptors, the ITAM includes acytoplasmic signaling sequence from CD3ζ (e.g., human CD3ζ).

In some embodiments of any of these single-chain chimeric antigenreceptors, any two neighboring domains can be separated by 1 amino acidsto about 500 amino acids (e.g., or any of the subranges of this rangedescribed herein).

In some embodiments, one or more amino acids between the extracellularhormone receptor ligand binding domain or the extracellularantigen-binding domain and the transmembrane domain is a sequence fromthe same endogenous single-chain polypeptide from which thetransmembrane domain is derived (e.g., a hinge sequence from human CD8α,e.g., SEQ ID NO: 112). In some embodiments, a sequence comprising SEQ IDNO: 42, 55, or 112 is positioned between the extracellular hormonereceptor ligand binding domain or the extracellular antigen-bindingdomain and the transmembrane domain. In some embodiments, one or moreamino acids between the extracellular antigen-binding domain or theextracellular hormone receptor ligand binding domain and thetransmembrane domain is or includes a hinge region sequence of anantibody such as, without limitation, a human antibody (e.g., IgG1,IgG2, IgG3, or IgG4). In some embodiments, one or more amino acidsbetween the extracellular antigen-binding domain or the extracellularhormone receptor ligand binding domain (e.g., any of the hormonereceptor ligand binding domain described herein or known in the art) andthe transmembrane domain is or comprises a linker sequence (e.g., anon-naturally occurring linker sequence). In some embodiments, one ormore amino acids between the extracellular antigen-binding domain andthe extracellular hormone receptor ligand binding domain is or comprisesa linker sequence (e.g., a non-naturally occurring linker sequence,e.g., GS or any of the other linker sequences described herein)).

In some embodiments of the single-chain chimeric antigen receptors,after the transmembrane domain, the next domain is a costimulatorydomain followed by an ITAM. In some embodiments of the single-chainchimeric antigen receptors, after the transmembrane domain, the nextdomain is an ITAM, followed by the costimulatory domain. In some ofthese embodiments, one or more amino acids between the transmembranedomain and the costimulatory domain or the ITAM is or includes asequence from the same endogenous single-chain polypeptide from whichthe costimulatory domain is derived or the ITAM is derived,respectively. In some of these embodiments, one or more amino acidsbetween the transmembrane domain and the costimulatory domain or theITAM is or includes a linker sequence (e.g., a non-naturally occurringlinker sequence). In some of these embodiments, one or more amino acidsbetween the the costimulatory domain and the ITAM is or includes alinker sequence (e.g., a non-naturally occurring linker sequence). Insome embodiments, one or more amino acids between the costimulatorydomain and the ITAM is or includes a sequence from the same endogenoussingle-chain polypeptide from which the costimulatory domain is derivedor the ITAM is derived.

In some embodiments, where two or more costimulatory domains areincluded in the single-chain chimeric antigen receptor, the two or morecostimulatory domains can be placed between the one or more ITAMs. Insome embodiments, where two or more ITAMs are included in thesingle-chain chimeric antigen receptor, the two or more ITAMs can beplaced between the one or more costimulatory molecules. In someembodiments of the single-chain chimeric antigen receptors describedherein, the costimulatory domains and ITAMs can alternate in the primaryamino acid sequence of the single-chain chimeric antigen receptor.

Some embodiments of any of the single-chain chimeric antigen receptorsdescribed herein can further include a dimerization domain and/or apeptide tag.

In some embodiments, when going in the N-terminal to C-terminaldirection, the single-chain chimeric antigen receptor comprises theextracellular antigen-binding domain, the extracellular hormone receptorligand binding domain, the transmembrane domain, the intracellularcostimulatory domain, and the ITAM. In some embodiments, when going inthe C-terminal to the N-terminal direction, the single-chain chimericantigen receptor comprises the extracellular antigen-binding domain, theextracellular hormone receptor ligand binding domain, the transmembranedomain, the intracellular costimulatory domain, and the ITAM. In someembodiments, when going in the N-terminal to the C-terminal direction,the single-chain chimeric antigen receptor comprises the extracellularhormone receptor ligand binding domain, the extracellularantigen-binding domain, the transmembrane domain, the intracellularcostimulatory domain, and the ITAM. In some embodiments, when going inthe C-terminal to the N-terminal direction, the single-chain chimericpolypeptide comprises the extracellular hormone receptor ligand bindingdomain, the extracellular antigen-binding domain, the transmembranedomain, the intracellular costimulatory domain, and the ITAM.

In some examples, a single-chain chimeric antigen receptor can comprisea sequence that is at least 80% (e.g., at least 82%, at least 84%, atleast 86%, at least 88%, at least 90%, at least 92%, at least 94%, atleast 96%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:98.

Multi-Chain Chimeric Antigen Receptor

Multi-Chain Chimeric Antigen Receptors—Intracellular Hormone ReceptorLBD

Also provided herein are multi-chain chimeric antigen receptors thatinclude at least one first polypeptide including: an extracellularantigen-binding domain (e.g., any of the antigen-binding domainsdescribed herein or known in the art), a transmembrane domain (e.g., anyof the transmembrane domains described herein or known in the art), anda hormone receptor ligand binding domain (e.g., any of the hormonereceptor ligand binding domains described herein or known in the art),where the transmembrane domain and the hormone receptor ligand bindingdomain directly abut each other or are separated by 1 to about 800 aminoacids (e.g., any of the subranges of this range described herein), andthe transmembrane domain and the hormone receptor ligand binding domainare not both present in the same (a single) endogenous single-chainpolypeptide. The antigen specifically bound by the antigen-bindingdomain in any of these multi-chain chimeric antigen receptors can be anyof the antigens described herein or known in the art. In someembodiments, the multi-chain chimeric antigen receptor only bindsspecifically to a single antigen (e.g., any of the exemplary antigensdescribed herein). In some embodiments, the multi-chain chimeric antigenreceptor binds specifically to two different antigens (e.g., anycombination of any of the exemplary antigens described herein).

In some embodiments of the multi-chain chimeric antigen receptors, theextracellular antigen-binding domain and the transmembrane domain in theat least one first polypeptide abut each other. In some embodiments ofthe multi-chain chimeric antigen receptors, 1 to about 500 amino acids(e.g., any of the subranges of this range described herein) are betweenthe extracellular antigen-binding domain and the transmembrane domain inthe at least one first polypeptide. In some embodiments, one or moreamino acids between the extracellular antigen-binding domain and thetransmembrane domain in the at least one first polypeptide is a sequencefrom the same endogenous single-chain polypeptide from which thetransmembrane domain is derived. In some embodiments, one or more aminoacids between the extracellular antigen-binding domain and thetransmembrane domain of the at least one first polypeptide is orincludes a hinge region sequence of human antibody (e.g., IgG1, IgG2,IgG3, or IgG4). In some embodiments, one or more amino acids between theextracellular antigen-binding domain and the transmembrane domain in theat least one first polypeptide is or includes a linker sequence (e.g., anon-naturally occurring linker sequence).

In some embodiments of these multi-chain chimeric antigen receptors, thetransmembrane domain abuts the hormone receptor ligand binding domain inthe at least one first polypeptide. In some embodiments of thesemulti-chain chimeric antigen receptors, the transmembrane domain and thehormone receptor ligand binding domain in the at least one firstpolypeptide are separated by 1 to about 800 amino acids (e.g., or any ofthe subranges of this range described herein). In some embodiments, theamino acids between the transmembrane domain and the hormone receptorligand binding domain in the at least one first polypeptide do notcomprise a domain. In other embodiments, the amino acids between thetransmembrane domain and the hormone receptor ligand binding domain inthe at least one first polypeptide includes one or more interveningdomain (e.g., one or more costimulatory domains (e.g., any of thecostimulatory domains described herein) and/or one or more ITAMs (e.g.,any of the ITAMs described herein)). In some embodiments, the aminoacids between the transmembrane domain and the hormone receptor ligandbinding domain in the at least one first polypeptide includes a sequencefrom the same endogenous transmembrane protein from which thetransmembrane domain is derived. In some embodiments, the amino acidsbetween the transmembrane domain and the hormone receptor ligand bindingdomain in the at least one first polypeptide include a linker sequence(e.g., a non-naturally occurring linker sequence).

In some embodiments of these multi-chain chimeric antigen receptors, theat least one first polypeptide can further include one or more (e.g.,two or three) of: a dimerizing domain (e.g., any dimerizing domain knownin the art), a costimulatory domain (e.g., any of the costimulatorydomains described herein), and/or a peptide tag (e.g., any peptide tagknown in the art).

Some embodiments of these multi-chain chimeric antigen receptors furtherinclude a second polypeptide that can include one or more (e.g., two,three, or four) of: a transmembrane domain (e.g., any of thetransmembrane domains described herein), a dimerizing domain (e.g., adimerizing domain that can interact with a dimerizing domain in the atleast one first polypeptide in a mammalian cell), one or morecostimulatory domains (e.g., any of the costimulatory domains describedherein), and one or more ITAM (e.g., any of the ITAMs described herein).As can be appreciated by those in the art, a pair or each pair ofneighboring domains in the second polypeptide can abut each other or canbe separated by 1 to about 800 amino acids (e.g., any of the subrangesof this range described herein). The one or more amino acids between apair of neighboring domains in the second polypeptide can be a sequencefrom an endogenous single-chain polypeptide from which a transmembrane,a costimulatory domain, or an ITAM present in the second polypeptide hasbeen derived.

Multi-Chain Chimeric Antigen Receptors—Extracellular Hormone ReceptorLBD

Also provided herein are multi-chain chimeric antigen receptors thatinclude at least one first polypeptide including: an extracellularantigen-binding domain (e.g., any of the antigen-binding domainsdescribed herein or known in the art), an extracellular hormone receptorligand binding domain (e.g., any of the hormone receptor ligand bindingdomains described herein or known in the art), and a transmembranedomain (e.g., any of the transmembrane domains described herein or knownin the art), where the transmembrane domain and the extracellularhormone receptor ligand binding domain directly abut each other or areseparated by 1 to about 800 amino acids (e.g., any of the subranges ofthis range described herein), and the transmembrane domain and thehormone receptor ligand binding domain are both not present in a singleendogenous single-chain polypeptide. The antigen specifically bound bythe antigen-binding domain in any of these multi-chain chimeric antigenreceptors can be any of the antigens described herein or known in theart. In some embodiments, the multi-chain chimeric antigen receptor onlybinds specifically to a single antigen (e.g., any of the exemplaryantigens described herein). In some embodiments, the multi-chainchimeric antigen receptor binds specifically to two different antigens(e.g., any combination of any of the exemplary antigens describedherein).

In some embodiments of the multi-chain chimeric antigen receptors, theextracellular hormone receptor ligand binding domain and thetransmembrane domain in the at least one first polypeptide abut eachother. In some embodiments of the multi-chain chimeric antigenreceptors, 1 to about 700 amino acids (e.g., any of the subranges ofthis range described herein) are between the extracellular hormonereceptor ligand binding domain and the transmembrane domain in the atleast one first polypeptide. In some embodiments, one or more aminoacids between the extracellular hormone receptor ligand binding domainand the transmembrane domain in the at least one first polypeptide is asequence from the same endogenous single-chain polypeptide from whichthe transmembrane domain is derived. In some embodiments, one or moreamino acids between the extracellular hormone receptor ligand bindingdomain and the transmembrane domain of the at least one firstpolypeptide is or includes a hinge region sequence of human antibody(e.g., IgG1, IgG2, IgG3, or IgG4). In some embodiments, one or moreamino acids between the extracellular hormone receptor ligand bindingdomain and the transmembrane domain in the at least one firstpolypeptide is or includes a linker sequence (e.g., a non-naturallyoccurring linker sequence). In some embodiments, one or more amino acidsbetween the extracellular hormone receptor ligand binding domain and theantigen-binding domain is or includes a linker sequence (e.g., anon-naturally occurring linker sequence).

In some embodiments of these multi-chain chimeric antigen receptors, thetransmembrane domain abuts the extracellular hormone receptor ligandbinding domain in the at least one first polypeptide. In someembodiments of these multi-chain chimeric antigen receptors, thetransmembrane domain and the extracellular hormone receptor ligandbinding domain in the at least one first polypeptide are separated by 1to about 800 amino acids (e.g., or any of the subranges of this rangedescribed herein). In some embodiments, the amino acids between thetransmembrane domain and the extracellular hormone receptor ligandbinding domain in the at least one first polypeptide do not comprise adomain.

In some embodiments, the amino acids between the transmembrane domainand the extracellular hormone receptor ligand binding domain in the atleast one first polypeptide includes one or more intervening domain(e.g., the antigen-binding domain). In some embodiments, the amino acidsbetween the transmembrane domain and the antigen-binding domain in theat least one first polypeptide includes a sequence from the sameendogenous transmembrane protein from which the transmembrane domain isderived. In some embodiments, the amino acids between the transmembranedomain and the antigen-binding domain in the at least one firstpolypeptide include a linker sequence (e.g., a non-naturally occurringlinker sequence). In some embodiments, one or more amino acids betweenthe antigen-binding domain and the transmembrane domain of the at leastone first polypeptide is or includes a hinge region sequence of humanantibody (e.g., IgG1, IgG2, IgG3, or IgG4). In some embodiments, one ormore amino acids between the antigen-binding domain and theextracellular hormone receptor ligand binding domain in the at least onefirst polypeptide is or includes a linker sequence (e.g., anon-naturally occurring linker sequence).

In some embodiments, when going in the N-terminal to C-terminaldirection, the at least one first polypeptide comprises theextracellular antigen-binding domain, the extracellular hormone receptorligand binding domain, and the transmembrane domain. In someembodiments, when going in the C-terminal to N-terminal direction, theat least one first polypeptide comprises the extracellularantigen-binding domain, the extracellular hormone receptor ligandbinding domain, and the transmembrane domain. In some embodiments, whengoing in the N-terminal to C-terminal direction, the at least one firstpolypeptide comprises the extracellular hormone receptor ligand bindingdomain, the extracellular antigen-binding domain, and the transmembranedomain. In some embodiments, when going in the C-terminal to N-terminaldirection, the at least one first polypeptide comprises theextracellular hormone receptor ligand binding domain, the extracellularantigen-binding domain, and the transmembrane domain.

In some embodiments of these multi-chain chimeric antigen receptors, theat least one first polypeptide can further include one or more (e.g.,two or three) of: a dimerizing domain (e.g., any dimerizing domain knownin the art), a costimulatory domain (e.g., any of the costimulatorydomains described herein), and/or a peptide tag (e.g., any peptide tagknown in the art).

Some embodiments of these multi-chain chimeric antigen receptors furtherinclude a second polypeptide that can include one or more (e.g., two,three, or four, in any order or combination) of: a transmembrane domain(e.g., any of the transmembrane domains described herein), a dimerizingdomain (e.g., a dimerizing domain that can interact with a dimerizingdomain in the at least one first polypeptide in a mammalian cell), oneor more costimulatory domains (e.g., any of the costimulatory domainsdescribed herein), and one or more ITAM (e.g., any of the ITAMsdescribed herein). As can be appreciated by those in the art, a pair oreach pair of neighboring domains in the second polypeptide can abut eachother or can be separated by 1 to about 800 amino acids (e.g., any ofthe subranges of this range described herein). The one or more aminoacids between a pair of neighboring domains in the second polypeptidecan be a sequence from an endogenous single-chain polypeptide from whicha transmembrane, a costimulatory domain, or an ITAM present in thesecond polypeptide has been derived. The one or more amino acids betweena pair of neighboring domains in the second polypeptide can be orinclude a linker sequence (e.g., a non-naturally occurring linkersequence).

As can be appreciated in the art, the two or more polypeptides presentin a multi-chain chimeric antigen receptor can associate via pair ofdomains that interact with each other (through dimerizing domains). Insome embodiments, the interaction between dimerizing domains can betriggered by the addition of a small molecule. In some embodiments, thetwo or more polypeptides present in a multi-meric chimeric antigenreceptor can associate through non-covalent interactions (e.g., betweenassociations between dimerizing domains). In some embodiments, the twoor more polypeptides present in a multi-meric chimeric antigen receptorcan be through a covalent interaction (e.g., through a disulfide bond,through an ester bond, through an amide bond, through a thioester bond,or a combination thereof).

Transmembrane Domains

In some embodiments, a single-chain chimeric polypeptide, a single-chainchimeric antigen receptor, or a multi-chain chimeric antigen receptorincludes a transmembrane domain, or portion thereof, from an endogenouspolypeptide, where the endogenous polypeptide is selected from the groupg of: an α chain of a T cell receptor, a β chain of the T cell receptor,a ζ chain of the T cell receptor, CD28 (also known as Tp44), CD3ε, CD3δ,CD3γ, CD33, CD37 (also known as GP52-40 or TSPAN26), CD64 (also known asFCGR1A), CD80 (also known as B7, B7-1, B7.1, BB1, CD28LG, CD28LG1, andLAB7), CD45 (also known as PTPRC, B220, CD45R, GP180, L-CA, LCA, LY5,T200, and protein tyrosine phosphatase, receptor type C), CD4, CD5 (alsoknown as LEU1 and T1), CD8α (also known as Leu2, MAL, and p32), CD9(also known as BTCC-1, DRAP-27, MIC3, MRP-1, TSPAN-29, and TSPAN29),CD16 (also known as FCGR3 and FCG3), CD22 (also known as SIGLEC-2 andSIGLEC2), CD86 (also known as B7-2, B7.2, B70, CD28LG2, and LAB72),CD134 (also known as TNFRSF4, ACT35, RP5-902P8.3, IMD16, OX40, TXGP1L,and tumor necrosis factor receptor superfamily member 4), CD137 (alsoknown as TNFRSF9, 4-1BB, CDw137, ILA, and tumor necrosis factor receptorsuperfamily member 9), CD27 (also known as S152, S152.LPFS2, T14,TNFRSF7, and Tp55), CD152 (also known as CTLA4, ALPS5, CELIAC3, CTLA-4,GRD4, GSE, IDDM12, and cytotoxic T-lymphocyte associated protein 4), PD1(also known as PDCD1, CD279, PD-1, SLEB2, hPD-1, hPD-1, hSLE1, andProgrammed cell death 1), ICOS (also known as AILIM, CD278, and CVID1),CD272 (also known as BTLA and BTLA1), CD30 (also known as TNFRSF8,D1S166E, and Ki-1), GITR (also known as TNFRSF18, RP5-902P8.2, AITR,CD357, and GITR-D), HVEM (also known as TNFRSF14, RP3-395M20.6, ATAR,CD270, HVEA, HVEM, LIGHTR, and TR2), DAP10, and CD154 (also known asCD40LG, CD40L, HIGM1, IGM, IMD3, T-BAM, TNFSF5, TRAP, gp39, hCD40L, andCD40 ligand). The letters “CD” is the previous sentence stand for“Cluster of Differentiation.” E.g., CD3 stands for “Cluster ofDifferentiation 3.” In some embodiments, a single-chain chimericpolypeptide, a single-chain chimeric antigen receptor, or a multi-chainchimeric antigen receptor includes a transmembrane domain, or portionthereof, from an endogenous mammalian (e.g., human) polypeptide (e.g., amammalian or human homolog of any of the polypeptides listed above).

Any transmembrane domain, or portion thereof, that serves to anchor anendogenous polypeptide in a lipid bilayer (e.g., plasma membrane) of amammalian cell is suitable for use in accordance with compositions andmethods disclosed herein. In some embodiments, a single-chain chimericpolypeptide, a single-chain chimeric antigen receptor, or a multi-chainchimeric antigen receptor includes a transmembrane domain, or portionthereof, from human CD28, e.g., Accession No. P01747, e.g., amino acids153 to 179 of SEQ ID NO: 1. In some embodiments, a single-chain chimericpolypeptide, a single-chain chimeric antigen receptor, or a multi-chainchimeric antigen receptor includes a transmembrane domain that is atleast 80% (e.g., at least 82%, at least 84%, at least 85%, at least 86%,at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, atleast 96%, at least 98%, or at least 99% identical) to amino acids 153to 179 of SEQ ID NO: 1, or a portion thereof.

In some embodiments, transmembrane domain can comprise a sequence atleast 80% (e.g., at least 82%, at least 84%, at least 86%, at least 88%,at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, atleast 99%, or 100% identical) to SEQ ID NO: 114 (or a portion thereof).

SEQ ID NO: 1 MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPG PTRKHYQPYAPPRDFAAYRS

In some embodiments, a single-chain chimeric polypeptide, a single-chainchimeric antigen receptor, or a multi-chain chimeric antigen receptorincludes a transmembrane domain, or portion thereof, from human CD3,e.g., Accession No. P20963, e.g., amino acids 31 to 51 of SEQ ID NO: 2.In some embodiments, a single-chain chimeric polypeptide, a single-chainchimeric antigen receptor, or a multi-chain chimeric antigen receptorincludes a transmembrane domain that is or includes a sequence that isat least 80% (e.g., at least 82%, at least 84%, at least 85%, at least86%, at least 88%, at least 90%, at least 92%, at least 94%, at least95%, at least 96%, at least 98%, or at least 99% identical) to aminoacids 31 to 51 of SEQ ID NO: 2.

SEQ ID NO: 2 MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK DTYDALHMQALPPR

In some embodiments, a single-chain chimeric polypeptide, a single-chainchimeric antigen receptor, or a multi-chain chimeric antigen receptorincludes a transmembrane domain, or portion thereof, of any one of SEQID Nos. 3-9.

(SEQ ID NO: 3) LGLLVAGVLVLLVSLGVAIHLCC; (SEQ ID NO: 4)VAAILGLGLVLGLLGPLAILLALYLL; (SEQ ID NO: 5) ALIVLGGVAGLLLFIGLGIFFCVRC;(SEQ ID NO: 6) LCYLLDGILFIYGVILTALFLRV; (SEQ ID NO: 7)WVLVVVGGVLACYSLLVTVAFIIFWV; (SEQ ID NO: 8) IYIWAPLAGTCGVLLLSLVITLYC; and(SEQ ID NO: 9) ALPAALAVISFLLGLGLGVACVLA.

In some embodiments, a single-chain chimeric polypeptide, a single-chainchimeric antigen receptor, or a multi-chain chimeric antigen receptorincludes a transmembrane domain that is or includes a sequence that isat least 80% (e.g., at least 82%, at least 84%, at least 85%, at least86%, at least 88%, at least 90%, at least 92%, at least 94%, at least95%, at least 96%, at least 98%, or at least 99% identical) to any oneof SEQ ID Nos. 3-9.

In some embodiments, a single-chain chimeric polypeptide, a single-chainchimeric antigen receptor, or a multi-chain chimeric antigen receptorincludes a transmembrane domain that is or includes a sequence that isat least 80% (e.g., at least 82%, at least 84%, at least 85%, at least86%, at least 88%, at least 90%, at least 92%, at least 94%, at least95%, at least 96%, at least 98%, or at least 99% identical) to SEQ IDNO: 43, 56, or 114.

As will be appreciated by those of ordinary skill in the art, certainendogenous polypeptides have two or more isoforms that differ at leastin their primary polypeptide sequence. A single-chain chimericpolypeptide, a single-chain chimeric antigen receptor, or a multi-chainchimeric antigen receptor disclosed herein can include a transmembranedomain that includes a sequence of amino acids from any isoform of anendogenous transmembrane protein (e.g., an endogenous mammalian, e.g.,human, transmembrane protein) including, e.g., an isoform (e.g., a humanisoform) of: an α chain of a T cell receptor, a β chain of the T cellreceptor, a ζ chain of the T cell receptor, CD28, CD3ε, CD3δ, CD3γ,CD33, CD37, CD64, CD80, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD86,CD134, CD137, CD27, CD152, PD1, or CD154.

In some embodiments, a transmembrane domain, or portion thereof, of asingle-chain chimeric polypeptide, a single-chain chimeric antigenreceptor, or a multi-chain chimeric antigen receptor includes a sequenceof amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to thetransmembrane domains from one or more of the following endogenousmammalian (e.g., human) transmembrane proteins: an α chain of a T cellreceptor, a β chain of the T cell receptor, a ζ chain of the T cellreceptor, CD28, CD3ε, CD3δ, CD3γ, CD33, CD37, CD64, CD80, CD45, CD4,CD5, CD8α, CD9, CD16, CD22, CD86, CD134, CD137, CD27, CD152, PD1, orCD154. In some embodiments, a transmembrane domain, or portion thereof,of a single-chain chimeric polypeptide, a single-chain chimeric antigenreceptor, or a multi-chain chimeric antigen receptor includes a sequenceof amino acids having one or more amino acid substitutions, deletions,or additions as compared to the transmembrane domain of an endogenousmammalian (e.g., human) transmembrane protein: an a chain of a T cellreceptor, a β chain of the T cell receptor, a ζ chain of the T cellreceptor, CD28, CD3ε, CD3δ, CD3γ, CD33, CD37, CD64, CD80, CD45, CD4,CD5, CD8α, CD9, CD16, CD22, CD86, CD134, CD137, CD27, CD152, PD1, orCD154.

In some embodiments, a single-chain chimeric polypeptide, a single-chainchimeric antigen receptor, or a multi-chain chimeric antigen receptorincludes a synthetic transmembrane domain. In some cases, a synthetictransmembrane domain can include predominantly hydrophobic residues suchas, without limitation, leucine and valine. In some embodiments, asynthetic transmembrane domain includes a triplet of phenylalanine,tryptophan, and valine at each end of the domain.

In some embodiments, a single-chain chimeric polypeptide, a single-chainchimeric antigen receptor, or a multi-chain chimeric antigen receptorincludes a transmembrane domain that is a chimeric transmembrane domainhaving portions of a transmembrane domain from two or more endogenousmammalian (e.g., human) transmembrane polypeptides such as, withoutlimitation, an α chain of a T cell receptor, a β chain of the T cellreceptor, a ζ chain of the T cell receptor, CD28, CD3ε, CD3δ, CD3γ,CD33, CD37, CD64, CD80, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD86,CD134, CD137, CD27, CD152, PD1, and CD154, such that the two or moreportions of transmembrane domains together constitute a functionaltransmembrane domain. In some embodiments, such a portion of a chimerictransmembrane domain can include one or more amino acid substitutions,deletions, or additions as compared to a corresponding portion of a wildtype transmembrane domain.

A transmembrane domain can include one, two, three, four, five, six,seven, eight, nine, or ten contiguous amino acid sequences that eachtraverse a lipid bilayer when present in the corresponding endogenouspolypeptide when expressed in a mammalian cell. As is known in the art,a transmembrane domain can, e.g., include at least one (e.g., two,three, four, five, six, seven, eight, nine, or ten) contiguous aminoacid sequence (that traverses a lipid bilayer when present in thecorresponding endogenous polypeptide when expressed in a mammalian cell)that has α-helical secondary structure in the lipid bilayer. In someembodiments, a transmembrane domain can include two or more contiguousamino acid sequences (that each traverse a lipid bilayer when present inthe corresponding endogenous polypeptide when expressed in a mammaliancell) that form a β-barrel secondary structure in the lipid bilayer.Additional examples and features of transmembrane domains are known inthe art.

Hormone Receptor Ligand Binding Domains

In some embodiments, the hormone receptor ligand binding domain is orcomprises an estrogen receptor ligand binding domain, or a fragmentthereof. Additional examples of hormone receptor ligand binding domainsthat can be used in any of the polypeptides described herein (e.g.,single-chain chimeric polypeptide, single-chain chimeric antigenreceptor, or multi-chain chimeric antigen receptor) include the ligandbinding domains of, e.g., an estrogen receptor, an ecdysone receptor, aPPARγ receptor, a glucocorticoid receptor, an androgen receptor, athyroid hormone receptor, a mineralocorticoid receptor, a progesteronereceptor, a vitamin D receptor, a PPAR receptorα, a PPARβ/δ receptor, apregnane X receptor, a liver X receptor, a farnesoid X receptor, aretinoid X receptor, a RAR-related orphan receptor, and a retinoic acidreceptor.

A non-limiting example of a human estrogen receptor ligand bindingdomain is amino acids 10 of 247 of SEQ ID NO: 10. In some embodiments, ahormone receptor ligand binding domain is or include a sequence that isat least 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to amino acids 10 to 247 of SEQ ID NO: 10.

(Human) SEQ ID NO: 10 1 skknslalsl tadqmvsall daeppilyse ydptrpfseasmmglltnla drelvhminw 61 akrvpgfvdl tlhdqvhlle cawleilmig lvwrsmehpgkllfapnlll drnqgkcveg 121 mveifdmlla tssrfrmmnl qgeefvclks iillnsgvytflsstlksle ekdhihrvld 181 kitdtlihlm akagltlqqq hqrlaqllli lshirhmsnkgmehlysmkc knvvplydll 241 lemldahrlh

Another non-limiting example of an estrogen receptor ligand bindingdomain is amino acids 4 to 241 of SEQ ID NO: 11. In some embodiments, ahormone receptor ligand binding domain is or include a sequence that isat least 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to amino acids 4 to 241 of SEQ ID NO: 11.

(Human) SEQ ID NO: 11 1 alsltadqmv salldaeppi lyseydptrp fseasmmglltnladrelvh minwakrvpg 61 fvdltlhdqv hllecawlei lmiglvwrsm ehpgkllfapnllldrnqgk cvegmveifd 121 mllatssrfr mmnlqgeefv clksiillns gvytflsstlksleekdhih rvldkitdtl 181 ihlmakaglt lqqqhqrlaq lllilshirh msnkgmehlysmkcknvvpl ydlllemlda 241 hrlhapts

In some embodiments, the estrogen receptor ligand binding domain has awildtype sequence, except that it comprises one or more (e.g., two,three, four, five, or six) amino acid substitutions. In someembodiments, the estrogen receptor ligand binding domain has a wildtypesequence, except that it comprises an amino acid substitution(s) at oneor both of amino acid position 521 (e.g., a G521R substitution) and 537(e.g., a G537F substitution) (each numbered relative to the full-lengthwildtype sequence of human estrogen receptor, e.g., SEQ ID NO: 119below).

(Human) SEQ ID NO: 119 1 mtmtlhtkas gmallhqiqg neleplnrpq lkiplerplgevyldsskpa vynypegaay 61 efnaaaaana qvygqtglpy gpgseaaafg snglggfpplnsvspsplml lhpppqlspf 121 lqphgqqvpy ylenepsgyt vreagppafy rpnsdnrrqggrerlastnd kgsmamesak 181 etrycavcnd yasgyhygvw scegckaffk rsiqghndymcpatnqctid knrrkscqac 241 rlrkcyevgm mkggirkdrr ggrmlkhkrq rddgegrgevgsagdmraan lwpsplmikr 301 skknslalsl tadqmvsall daeppilyse ydptrpfseasmmglltnla drelvhminw 361 akrvpgfvdl tlhdqvhlle cawleilmig lvwrsmehpgkllfapnlll drnqgkcveg 421 mveifdmlla tssrfrmmnl qgeefvclks iillnsgvytflsstlksle ekdhihrvld 481 kitdtlihlm akagltlqqq hqrlaqllli lshirhmsnkgmehlysmkc knvvplydll 541 lemldahrlh aptsrggasv eetdqshlat agstsshslqkyyitgeaeg fpatv

Non-limiting examples of an estrogen receptor ligand binding domain caninclude a sequence at least 80% (e.g., at least 82%, at least 84%, atleast 86%, at least 88%, at least 90%, at least 92%, at least 94%, atleast 96%, at least 98%, at least 99% or 100%) identical to: SEQ ID NO:110 (or a fragment thereof).

Additional non-limiting examples of a hormone receptor ligand bindingdomain is or includes a sequence at least 80% (e.g., at least 82%, atleast 84%, at least 86%, at least 88%, at least 90%, at least 92%, atleast 94%, at least 96%, at least 98%, at least 99% or 100%) identicalto SEQ ID NO: 46, 59, or 110.

Another non-limiting example of an estrogen receptor ligand bindingdomain is amino acids 314 to 551 of SEQ ID NO: 88. In some embodiments,a hormone receptor ligand binding domain is or include a sequence thatis at least 80%, at least 81%, at least 82%, at least 83%, at least 84%,at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to amino acids 314 to 551 of SEQ ID NO: 88.

(Mouse) SEQ ID NO: 88 1 mtmtlhtkas gmallhqiqg neleplnrpq lkmpmeralgevyvdnskpt vfnypegaay 61 efnaaaaaaa aasapvygqs giaygpgsea aafsanslgafpqlnsvsps plmllhpppq 121 lspflhphgq qvpyylenep sayavrdtgp pafyrsnsdnrrqngrerls ssnekgnmim 181 esaketryca vcndyasgyh ygvwscegck affkrsiqghndymcpatnq ctidknrrks 241 cqacrlrkcy evgmmkggir kdrrggrmlk hkrqrddlegrnemgasgdm raanlwpspl 301 vikhtkknsp alsltadqmv salldaeppm iyseydpsrpfseasmmgll tnladrelvh 361 minwakrvpg fgdlnlhdqv hllecawlei lmiglvwrsmehpgkllfap nllldrnqgk 421 cvegmveifd mllatssrfr mmnlqgeefv clksiillnsgvytflsstl ksleekdhih 481 rvldkitdtl ihlmakaglt lqqqhrrlaq lllilshirhmsnkgmehly nmkcknvvpl 541 ydlllemlda hrlhapasrm gvppeepsqt qlattsstsahslqtyyipp eaegfpnti

Another non-limiting example of an estrogen receptor ligand bindingdomain is amino acids 315 to 552 of SEQ ID NO: 89. In some embodiments,a hormone receptor ligand binding domain is or include a sequence thatis at least 80%, at least 81%, at least 82%, at least 83%, at least 84%,at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to amino acids 315 to 552 of SEQ ID NO: 89.

(Rat) SEQ ID NO: 89 1 mtmtlhtkas gmallhqiqg neleplnrpq lkmpmeralgevyvdnskpa vfnypegaay 61 efnaaaaaaa agasapvygq ssitygpgse aaafganslgafpqlnsvsp splmllhppp 121 hvspflhphg hqvpyylene psayavrdtg ppafyrsnsdnrrqngrerl ssssekgnmi 181 mesaketryc avcndyasgy hygvwscegc kaffkrsiqghndymcpatn qctidknrrk 241 scqacrlrkc yevgmmkggi rkdrrggrml khkrqrddlegrnemgtsgd mraanlwpsp 301 lvikhtkkns palsltadqm vsalldaepp liyseydpsrpfseasmmgl ltnladrelv 361 hminwakrvp gfgdlnlhdq vhllecawle ilmiglvwrsmehpgkllfa pnllldrnqg 421 kcvegmveif dmllatssrf rmmnlqgeef vclksiillnsgvytflsst lksleekdhi 481 hrvldkindt lihlmakagl tlqqqhrrla qlllilshirhmsnkgmehl ynmkcknvvp 541 lydlllemld ahrlhapasr mgvppeepsq sqltttsstsahslqtyyip peaegfpnti

Another non-limiting example of an estrogen receptor ligand bindingdomain is amino acids 310 to 547 of SEQ ID NO: 90. In some embodiments,a hormone receptor ligand binding domain is or include a sequence thatis at least 80%, at least 81%, at least 82%, at least 83%, at least 84%,at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to amino acids 310 to 547 of SEQ ID NO: 90.

(Baboon) SEQ ID NO: 90 1 mtmtlhtkas gmallhqiqg neleplnrpq lkiplerplgevyvdsskpa vysypegaay 61 efnaaaaana qvygqtglpy gpgseaaafg snglggfpplnsvspsplml lhpppqlspf 121 lqphgqqvpy ylenepsgyt vreagppafy rpnsdnrrqggrerlastnd kgsmamesak 181 etrycavcnd yasgyhygvw scegckaffk rsiqghndymcpatnqctid knrrkscqac 241 rlrkcyevgm mkggirkdrr ggrmlkhkrq rddgegrgevgsagdmraan lwpsplmikh 301 skknspalsl tadqmvsall daeppilyse ydptrpfseasmmglltnla drelvhminw 361 akrvpgfvdl tlhdqvhlle cawleilmig lvwrsmehpgkllfapnlll drnqgkcveg 421 mveifdmlla tssrfrmmnl qgeefvclks iillnsgvytflsstlksle ekdhihrvld 481 kitdtlihlm akagltlqqq hrrlaqllli lshirhmsnkgmehlysmkc knvvplydll 541 lemldahrlh aptsrggapm eetdqshlat agstsshslqkyyitgdaeg fpatv

In some embodiments, the hormone receptor ligand binding domain is orcomprises a progesterone receptor ligand binding domain, or a fragmentthereof. A non-limiting example of a human progesterone receptor ligandbinding domain is amino acids 11 to 258 of SEQ ID NO: 12. In someembodiments, a hormone receptor ligand binding domain is or include asequence that is at least 80%, at least 81%, at least 82%, at least 83%,at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% identical to amino acids 11 to 258 of SEQ ID NO: 12.

(Human) SEQ ID NO: 12 1 spgqdiqlip plinllmsie pdviyaghdn tkpdtssslltslnqlgerq llsvvkwsks 61 lpgfrnlhid dqitliqysw mslmvfglgw rsykhvsgqmlyfapdliln eqrmkessfy 121 slcltmwqip qefvklqvsq eeflcmkvll llntipleglrsqtqfeemr ssyirelika 181 iglrqkgvvs ssqrfyqltk lldnlhdlvk qlhlyclntfiqsralsvef pemmseviaa 241 qlpkilagmv kpllfhkk

Another non-limiting example of a progesterone receptor ligand bindingdomain is amino acids 14 to 261 of SEQ ID NO: 13. In some embodiments, ahormone receptor ligand binding domain is or include a sequence that isat least 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to amino acids 14 to 261 of SEQ ID NO: 13.

(Human) SEQ ID NO: 13 1 ftfspgqdiq lipplinllm siepdviyag hdntkpdtssslltslnqlg erqllsvvkw 61 skslpgfrnl hiddqitliq yswmslmvfg lgwrsykhvsgqmlyfapdl ilneqrmkes 121 sfyslcltmw qipqefvklq vsqeeflcmk vllllntipleglrsqtqfe emrssyirel 181 ikaiglrqkg vvsssqrfyq ltklldnlhd lvkqlhlyclntfiqsrals vefpemmsev 241 iaaqlpkila gmvkpllfhk k

Another non-limiting example of a progesterone receptor ligand bindingdomain is amino acids 679 to 926 of SEQ ID NO: 91. In some embodiments,a hormone receptor ligand binding domain is or include a sequence thatis at least 80%, at least 81%, at least 82%, at least 83%, at least 84%,at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to amino acids 679 to 926 of SEQ ID NO: 91.

(Mouse) SEQ ID NO: 91 1 mtelqakdpq vlhtsgasps pphigsplla rldsgpfqgsqhsdvssvvs pipisldgll 61 fprscrgpel pdgktgdqqs lsdvegafsg veathreggrnsrapekdsr lldsvldsll 121 tpsgteqsha sppaceaits wclfgpelpe dprsvpatkgllsplmsrpe ikagdssgtg 181 agqkvlpkgl spprqlllpt sgsahwpgag vkpspqpaageveedsglet egsaapllks 241 kpralegtgs gggvaanaas aapggvtlvp kedsrfsaprvsleqdspia pgrsplattv 301 vdfihvpilp lnhallaart rqllegdsyd ggataqgpfapprgspsaps ppvpcgdfpd 361 ctyplegdpk edvfplygdf qtpglkikee eegadaavrsprpylsagas sstfpdfpla 421 papqrapssr pgeaavaggp ssaavspass sgsalecilykaegapptqg sfaplpckpp 481 aagscllprd slpaapataa apaiyqplgl nglpqlgyqaavlkdslpqv yppylnylrp 541 dseasqspqy gfdslpqkic licgdeasgc hygvltcgsckvffkrameg qhnylcagrn 601 dcivdkirrk ncpacrlrkc cqagmvlggr kfkkfnkvrvmrtldgvalp qsvglpnesq 661 algqritfsp nqeiqlvppl inllmsiepd vvyaghdntkpdtsssllts lnqlgerqll 721 svvkwskslp gfrnlhiddq itliqyswms lmvfglgwrsykhvsgqmly fapdlilneq 781 rmkelsfysl cltmwqipqe fvklqvthee flcmkvllllntipleglrs qsqfeemrss 841 yirelikaig lrqkgvvpss qrfyqltkll dslhdlvkqlhlyclntfiq srtlavefpe 901 mmseviaaql pkilagmvkp llfhkk

Another non-limiting example of a progesterone receptor ligand bindingdomain is amino acids 676 to 923 of SEQ ID NO: 92. In some embodiments,a hormone receptor ligand binding domain is or include a sequence thatis at least 80%, at least 81%, at least 82%, at least 83%, at least 84%,at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to amino acids 676 to 923 of SEQ ID NO: 92.

(Rat) SEQ ID NO: 92 1 mtelqakdpr tlhtsgaaps pthvgsplla rldpdpfqgsqhsdassvvs pipisldrll 61 fsrscqaqel pdektqnqqs lsdvegafsg veasrrrsrnprapekdsrl ldsvldtlla 121 psgpeqsqts ppaceaitsw clfgpelped prsvpatkgllsplmsrpes kagdssgtga 181 gqkvlpkavs pprqlllpts gsahwpgagv kpsqqpatveveedgglete gsagpllksk 241 pralegmcsg ggvtanapga apggvtlvpk edsrfsaprvsleqdapvap grsplattvv 301 dfihvpilpl nhallaartr qllegdsydg gaaaqvpfapprgspsapsp pvpcgdfpdc 361 typpegdpke dgfpvygefq ppglkikeee egteaasrsprpyllagasa atfpdfplpp 421 rpprappsrp geaavaapsa avspvsssgs alecilykaegapptqgsfa plpckppaas 481 scllprdslp aaptssaapa iypplglngl pqlgyqaavlkdslpqvypp ylnylrpdse 541 asqspqygfd slpqkiclic gdeasgchyg vltcgsckvffkramegqhn ylcagrndci 601 vdkirrkncp acrlrkccqa gmvlggrkfk kfnkvrvmraldgvalpqsv afpnesqtlg 661 qritfspnqe iqlvpplinl lmsiepdvvy aghdntkpdtssslltslnq lgerqllsvv 721 kwskslpgfr nlhiddqitl iqyswmslmv fglgwrsykhvsgqmlyfap dlilneqrmk 781 elsfyslclt mwqipqefvk lqvtheeflc mkvllllntipleglrsqsq feemrssyir 841 elikaiglrq kgvvpssqrf yqltklldsl hdlvkqlhlyclntfiqsra lavefpemms 901 eviaaqlpki lagmvkpllf hkk

Another non-limiting example of a progesterone receptor ligand bindingdomain is amino acids 686 to 933 of SEQ ID NO: 93. In some embodiments,a hormone receptor ligand binding domain is or include a sequence thatis at least 80%, at least 81%, at least 82%, at least 83%, at least 84%,at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to amino acids 686 to 933 of SEQ ID NO: 93.

(Baboon) SEQ ID NO: 93 1 mtelkakgpr aphvaggpps pevgspllcr paagpfqgsqtsdtlpevsa ipisldgllf 61 prpcqgqdpl dektqdqqsl sdvegaysra eatrgtggsssrppekdsgl lhsvldtlla 121 psgpgqsqps ppacevtssw clfgpelped ppaapatqgvlsplmsrsgc kagdssgtaa 181 ahkvlprgls psrqlllpas gsphwsgapv kpspqpaaveveeedgsese esagpllkgk 241 pralggaaag ggaaavppga aaggvalvpk edsrfsaprvalveqdapma pgrsplattt 301 mdfthvpilp lshallaart rqlleeesyd ggagaasafapprsspsass tpvavgdfpd 361 cayppdadpk ddayplygdf qppalkikee eegaevsarsprsylvagan paafpdfplg 421 pppplpprap psrpgeaavt aapagasvss asssgstlecilykaegapp qqgpfapppc 481 kapgaggcll prdglpstsa saaaagaapa lypalglnglpqlgyqaavl keglqqvypp 541 ylnylrpdse asqspqysfe slpqkiclic gdeasgchygvltcgsckvf fkramegqhn 601 ylcagrndci vdkirrkncp acrlrkccqa gmvlggrkfkkfnkvrvmra ldavalpqpv 661 gipnesqals qrftfppgqd iqlipplinl lvsiepdviyaghdnskpdt ssslltslnq 721 lgerqllsvv kwskllpgfr nlhiddqitl iqyswmslmvfglgwrsykh vsgqmlyfap 781 dlilneqrmk essfyslclt mwqipqefvk lqvsqeeflcmkvllllnti pleglrsqtq 841 feemrssyir elikaiglrq kgvvsssqrf yqltklldnlhdlvkqlhly clntfiqsra 901 lsvefpemms eviaaqlpki lagmvkpllf hkk

In some embodiments, the hormone receptor ligand binding domain is orcomprises an androgen receptor ligand binding domain, or a fragmentthereof. A non-limiting example of a human androgen receptor ligandbinding domain is amino acids 2 of 247 of SEQ ID NO: 14. In someembodiments, a hormone receptor ligand binding domain is or include asequence that is at least 80%, at least 81%, at least 82%, at least 83%,at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% identical to amino acids 2 to 247 of SEQ ID NO: 14.

(Human) SEQ ID NO: 14 1 piflnvleai epgvvcaghd nnqpdsfaal lsslnelgerqlvhvvkwak alpgfrnlhv 61 ddqmaviqys wmglmvfamg wrsftnvnsr mlyfapdlvfneyrmhksrm ysqcvrmrhl 121 sqefgwlqit pqeflcmkal llfsiipvdg lknqkffdelrmnyikeldr iiackrknpt 181 scsrrfyqlt klldsvqpia relhqftfdl likshmvsvdfpemmaeiis vqvpkilsgk 241 vkpiyfhtq

Another non-limiting example of an androgen receptor ligand bindingdomain is amino acids 34 to 279 of SEQ ID NO: 15. In some embodiments, ahormone receptor ligand binding domain is or include a sequence that isat least 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to amino acids 34 to 279 of SEQ ID NO: 15.

(Human) SEQ ID NO: 15 1 sleegeasst tspteettqk ltvshiegye cqpiflnvleaiepgvvcag hdnnqpdsfa 61 allsslnelg erqlvhvvkw akalpgfrnl hvddqmaviqyswmglmvfa mgwrsftnvn 121 srmlyfapdl vfneyrmhks rmysqcvrmr hlsqefgwlqitpqeflcmk alllfsiipv 181 dglknqkffd elrmnyikel driiackrkn ptscsrrfyqltklldsvqp iarelhqftf 241 dllikshmvs vdfpemmaei isvqvpkils gkvkpiyfhtqeg

Another non-limiting example of an androgen receptor ligand bindingdomain is amino acids 19 to 264 of SEQ ID NO: 16. In some embodiments, ahormone receptor ligand binding domain is or include a sequence that isat least 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to amino acids 19 to 264 of SEQ ID NO: 16.

(Human) SEQ ID NO: 16 1 ettqkltvsh iegyecqpif lnvleaiepg vvcaghdnnqpdsfaallss lnelgerqlv 61 hvvkwakalp gfrnlhvddq maviqyswmg lmvfamgwrsftnvnsrmly fapdlvfney 121 rmhksrmysq cvrmrhlsqe fgwlqitpqe flcmkalllfsiipvdglkn qkffdelrmn 181 yikeldriia ckrknptscs rrfyqltkll dsvqpiarelhqftfdllik shmvsvdfpe 241 mmaeiisvqv pkilsgkvkp iyfhtq

Another non-limiting example of an androgen receptor ligand bindingdomain is amino acids 652 to 897 of SEQ ID NO: 94. In some embodiments,a hormone receptor ligand binding domain is or include a sequence thatis at least 80%, at least 81%, at least 82%, at least 83%, at least 84%,at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to amino acids 652 to 897 of SEQ ID NO: 94.

(Mouse) SEQ ID NO: 94 1 mevqlglgrv yprppsktyr gafqnlfqsv reaiqnpgprhpeaaniapp gaclqqrqet 61 sprrrrrqqh tedgspqahi rgptgylale eeqqpsqqqaaseghpessc lpepgaatap 121 gkglpqqppa ppdqddsaap stlsllgptf pglsscsadikdilneagtm qllqqqqqqq 181 qhqqqhqqhq qqqevisegs sarareatga pssskdsylggnstisdsak elckavsvsm 241 glgvealehl spgeqlrgdc myasllggpp avrptpcaplpeckglplde gpgksteeta 301 eyssfkggya kglegeslgc sgsseagssg tleipsslslyksgaldeaa ayqnrdyynf 361 plalsgpphp pppthphari klenpldygs awaaaaaqcrygdlgslhgg svagpstgsp 421 pattssswht lftaeegqly gpgggggsss psdagpvapygytrppqglt sqesdysase 481 vwypggvvnr vpypspncvk semgpwmeny sgpygdmrldstrdhvlpid yyfppqktcl 541 icgdeasgch ygaltcgsck vffkraaegk qkylcasrndctidkfrrkn cpscrlrkcy 601 eagmtlgark lkklgnlklq eegensnags ptedpsqkmtvshiegyecq piflnvleai 661 epgvvcaghd nnqpdsfaal lsslnelger qlvhvvkwakalpgfrnlhv ddqmaviqys 721 wmglmvfamg wrsftnvnsr mlyfapdlvf neyrmhksrmysqcvrmrhl sqefgwlqit 781 pqeflcmkal llfsiipvdg lknqkffdel rmnyikeldriiackrknpt scsrrfyqlt 841 klldsvqpia relhqftfdl likshmvsvd fpemmaeiisvqvpkilsgk vkpiyfhtq

Another non-limiting example of an androgen receptor ligand bindingdomain is amino acids 655 to 900 of SEQ ID NO: 95. In some embodiments,a hormone receptor ligand binding domain is or include a sequence thatis at least 80%, at least 81%, at least 82%, at least 83%, at least 84%,at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to amino acids 655 to 900 of SEQ ID NO: 95.

(Rat) SEQ ID NO: 95 1 mevqlglgrv yprppsktyr gafqnlfqsv reaiqnpgprhpeaasiapp gaclqqrqet 61 sprrrrrqqh pedgspqahi rgttgylale eeqqpsqqqsaseghpesgc lpepgaatap 121 gkglpqqppa ppdqddsaap stlsllgptf pglsscsadikdilseagtm qllqqqqqqq 181 qqqqqqqqqq qqqqqevise gsssvrarea tgapssskdsylggnstisd sakelckavs 241 vsmglgveal ehlspgeqlr gdcmyasllg gppavrptpcaplaeckgls ldegpgkgte 301 etaeyssfkg gyakgleges lgcsgsseag ssgtleipsslslyksgavd eaaayqnrdy 361 ynfplalsgp phppppthph ariklenpsd ygsawaaaaaqcrygdlasl hggsvagpst 421 gsppatasss whtlftaeeg qlygpggggg ssspsdagpvapygytrppq glasqegdfs 481 asevwypggv vnrvpypsps cvksemgpwm enysgpygdmrldstrdhvl pidyyfppqk 541 tclicgdeas gchygaltcg sckvffkraa egkqkylcasrndctidkfr rkncpscrlr 601 kcyeagmtlg arklkklgnl klqeegenss agsptedpsqkmtvshiegy ecqpiflnvl 661 eaiepgvvca ghdnnqpdsf aallsslnel gerqlvhvvkwakalpgfrn lhvddqmavi 721 qyswmglmvf amgwrsftnv nsrmlyfapd lvfneyrmhksrmysqcvrm rhlsqefgwl 781 qitpqeflcm kalllfsiip vdglknqkff delrmnyikeldriiackrk nptscsrrfy 841 qltklldsvq piarelhqft fdllikshmv svdfpemmaeiisvqvpkil sgkvkpiyfh 901 tq

Another non-limiting example of an androgen receptor ligand bindingdomain is amino acids 648 to 893 of SEQ ID NO: 96. In some embodiments,a hormone receptor ligand binding domain is or include a sequence thatis at least 80%, at least 81%, at least 82%, at least 83%, at least 84%,at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to amino acids 648 to 893 of SEQ ID NO: 96.

(Baboon) SEQ ID NO: 96 1 mevqlglgrv yprppsktyr gafqnlfqsv reviqnpgprhpeaasaapp gaslqqqqqq 61 qqqetsprqq qqqqgedgsp qahrrgptgy lvldeeqqpsqpqsapechp ergcvpepga 121 avaagkglpq qlpappdedd saapstlsll gptfpglsscsadlkdilse astmqllqqq 181 qqeavsegss sgrareasga ptsskdnylg gtstisdsakelckavsvsm glgvealehl 241 spgeqlrgdc myapvlgvpp avrptpcapl aeckgsllddsagkstedta eyspfkggyt 301 kglegeslgc sgsaaagssg tlelpstlsl yksgaldeaaayqsrdyynf plalagpppp 361 pppphphari klenpldygs awaaaaaqcr ygelaslhgagaagpgsgsp saaassswht 421 lftaeegqly gpcggggggg ggggggagea gavapygytrppqglagqeg dftapdvwyp 481 ggmvsrvpyp sptcvksemg pwmdsysgpy gdmrletardhvlpidyyfp pqktclicgd 541 easgchygal tcgsckvffk raaegkqkyl casrndctidkfrrkncpsc rlrkcyeagm 601 tlgarklkkl gnlklqeege assttsptee taqkltvshiegyecqpifl nvleaiepgv 661 vcaghdnnqp dsfaallssl nelgerqlvh vvkwakalpgfrnlhvddqm aviqyswmgl 721 mvfamgwrsf tnvnsrmlyf apdlvfneyr mhksrmysqcvrmrhlsqef gwlqitpqef 781 lcmkalllfs iipvdglknq kffdelrmny ikeldriiackrknptscsr rfyqltklld 841 svqpiarelh qftfdlliks hmvsvdfpem maeiisvqvpkilsgkvkpi yfhtq

Another non-limiting example of a ligand binding domain is SEQ ID NO:28. In some embodiments, a hormone receptor ligand binding domain is orincludes a sequence that is at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identical to the amino acid sequence of SEQID NO: 28.

(Human) SEQ ID NO: 28 1 drrggrmlkh krqrddgegr gevgsagdmr aanlwpsplmikrskknsla lsltadqmvs 61 alldaeppil yseydptrpf seasmmgllt nladrelvhminwakrvpgf vdltlhdqvh 121 llecawleil miglvwrsme hpgkllfapn llldrnqgkcvegmveifdm llatssrfrm 181 mnlqgeefvc lksiillnsg vytflsstlk sleekdhihrvldkitdtli hlmakagltl 241 qqqhqrlaql llilshirhm snkrmehlys mkcknvvplfdlllemldah rlhapts

Another non-limiting example of a ligand binding domain is SEQ ID NO:29. In some embodiments, a hormone receptor ligand binding domain is orincludes a sequence that is at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identical to the amino acid sequence of SEQID NO: 29.

(Human) SEQ ID NO: 29 1 gqdiqlippl inllmsiepd viyaghdntk pdtssslltslnqlgerqll svvkwskslp 61 gfrnlhiddq itliqyswms lmvfglgwrs ykhvsgqmlyfapdlilneq rmkessfysl 121 cltmwqipqe fvklqvsqee flcmkvllll ntipleglrsqtqfeemrss yirelikaig 181 lrqkgvvsss qrfyqltkll dnlhdlvkql hlyclntfiqsralsvefpe mmseviaaql 241 pkilagmvkp llfhkk

Another non-limiting example of a ligand binding domain is a ligandbinding domain of an ecdysone receptor. For example, a ligand bindingdomain of an ecdysone receptor can be amino acids 256 to 467 of SEQ IDNO: 74. In some embodiments, a hormone receptor ligand binding domain isor includes a sequence that is at least 80%, at least 81%, at least 82%,at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identical to amino acids 256 to 467 of SEQ IDNO: 74.

(Human) SEQ ID NO: 74 1 merdepppsg ggggggsagf leppaalppp prngfcqdelaeldpgtisv sddraeqrtc 61 licgdratgl hygiiscegc kgffkrsicn krvyrcsrdkncvmsrkqrn rcqycrllkc 121 lqmgmnrkai redgmpggrn ksigpvqise eeierimsgqefeeeanhws nhgdsdhssp 181 gnrasesnqp spgstlsssr svelngfmaf reqymgmsvpphyqyiphlf sysghspllp 241 qqarsldpqs yslihqllsa edleplgtpm liedgyavtqaelfallcrl adellfrqia 301 wikklpffce lsikdytcll sstwqelill ssltvyskqifgeladvtak yspsdeelhr 361 fsdegmevie rliylyhkfh qlkvsneeya cmkainflnqdirgltsasq leqlnkrywy 421 icqdfteyky thqpnrfpdl mmclpeiryi agkmvnvpleqlpllfkvvl hscktsvgke

Another non-limiting example of a ligand binding domain is a ligandbinding domain of a PPARγ receptor. For example, a ligand binding domainof a PPARγ receptor can be amino acids 237 to 504 of SEQ ID NO: 75. Insome embodiments, a hormone receptor ligand binding domain is orincludes a sequence that is at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identical to amino acids 237 to 504 of SEQ IDNO: 75.

(Human) SEQ ID NO: 75 1 mgetlgdspi dpesdsftdt lsanisqemt mvdtempfwptnfgissvdl svmedhshsf 61 dikpfttvdf ssistphyed ipftrtdpvv adykydlklqeyqsaikvep asppyysekt 121 qlynkpheep snslmaiecr vcgdkasgfh ygvhacegckgffrrtirlk liydrcdlnc 181 rihkksrnkc qycrfqkcla vgmshnairf grmpqaekekllaeissdid qlnpesadlr 241 alakhlydsy iksfpltkak arailtgktt dkspfviydmnslmmgedki kfkhitplqe 301 qskevairif qgcqfrsvea vqeiteyaks ipgfvnldlndqvtllkygv heiiytmlas 361 lmnkdgvlis egqgfmtref lkslrkpfgd fmepkfefavkfnaleldds dlaifiavii 421 lsgdrpglln vkpiediqdn llqalelqlk lnhpessqlfakllqkmtdl rqivtehvql 481 lqvikktetd mslhpllqei ykdly

Another non-limiting example of a ligand binding domain is a ligandbinding domain of a glucocorticoid receptor. For example, a ligandbinding domain of a glucocorticoid receptor can be amino acids 531 to777 of SEQ ID NO: 76. In some embodiments, a hormone receptor ligandbinding domain is or includes a sequence that is at least 80%, at least81%, at least 82%, at least 83%, at least 84%, at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% identical to aminoacids 531 to 777 of SEQ ID NO: 76.

(Human) SEQ ID NO: 76 1 mdskesltpg reenpssvla qergdvmdfy ktlrggatvkvsasspslav asqsdskqrr 61 llvdfpkgsv snaqqpdlsk avslsmglym getetkvmgndlgfpqqgqi slssgetdlk 121 lleesianln rstsvpenpk ssastavsaa ptekefpkthsdvsseqqhl kgqtgtnggn 181 vklyttdqst fdilqdlefs sgspgketne spwrsdllidencllsplag eddsfllegn 241 snedckplil pdtkpkikdn gdlvlsspsn vtlpqvktekedfielctpg vikqeklgtv 301 ycqasfpgan iignkmsais vhgvstsggq myhydmntaslsqqqdqkpi fnvippipvg 361 senwnrcqgs gddnltslgt lnfpgrtvfs ngysspsmrpdvssppssss tattgpppkl 421 clvcsdeasg chygvltcgs ckvffkrave gqhnylcagrndciidkirr kncpacryrk 481 clqagmnlea rktkkkikgi qqattgvsqe tsenpgnktivpatlpqltp tlvsllevie 541 pevlyagyds svpdstwrim ttlnmlggrq viaavkwakaipgfrnlhld dqmtllqysw 601 mflmafalgw rsyrqssanl lcfapdliin eqrmtlpcmydqckhmlyvs selhrlqvsy 661 eeylcmktll llssvpkdgl ksqelfdeir mtyikelgkaivkregnssq nwqrfyqltk 721 lldsmhevve nllnycfqtf ldktmsiefp emlaeiitnqipkysngnik kllfhqk

Another non-limiting example of a ligand binding domain is a ligandbinding domain of a thyroid hormone receptor. For example, a ligandbinding domain of a glucocorticoid receptor can be amino acids 162 to370 of SEQ ID NO: 77. In some embodiments, a hormone receptor ligandbinding domain is or includes a sequence that is at least 80%, at least81%, at least 82%, at least 83%, at least 84%, at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% identical to aminoacids 162 to 370 of SEQ ID NO: 77.

(Human) SEQ ID NO: 77 1 meqkpskvec gsdpeensar spdgkrkrkn gqcslktsmsgyipsyldkd eqcvvcgdka 61 tgyhyrcitc egckgffrrt iqknlhptys ckydsccvidkitrnqcqlc rfkkciavgm 121 amdlvlddsk rvakrklieq nrerrrkeem irslqqrpeptpeewdlihi ateahrstna 181 qgshwkqrrk flpddigqsp ivsmpdgdkv dleafseftkiitpaitrvv dfakklpmfs 241 elpcedqiil lkgccmeims lraavrydpe sdtltlsgemavkreqlkng glgvvsdaif 301 elgkslsafn lddtevallq avllmstdrs gllcvdkieksqeayllafe hyvnhrkhni 361 phfwpkllmk erevqssily kgaaaegrpg gslgvhpegqqllgmhvvqg pqvrqleqql 421 geagslqgpv lqhqspkspq qrllellhrs gilharavcgeddsseadsp ssseeepevc 481 edlagnaasp

Another non-limiting example of a ligand binding domain is a ligandbinding domain of a mineralocorticoid receptor. For example, a ligandbinding domain of a mineralocorticoid receptor can be amino acids 737 to984 of SEQ ID NO: 78. In some embodiments, a hormone receptor ligandbinding domain is or includes a sequence that is at least 80%, at least81%, at least 82%, at least 83%, at least 84%, at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% identical to aminoacids 737 to 984 of SEQ ID NO: 78.

(Human) SEQ ID NO: 78 1 metkgyhslp egldmerrwg qvsqaverss lgptertdennymeivnvsc vsgaipnnst 61 qgsskekqel lpclqqdnnr pgiltsdikt eleskelsatvaesmglymd svrdadysye 121 qqnqqgsmsp akiyqnveql vkfykgnghr pstlscvntplrsfmsdsgs svnggvmrai 181 vkspimchek spsvcsplnm tssvcspagi nsvssttasfgsfpvhspit qgtpltcspn 241 aenrgsrshs pahasnvgsp lssplssmks sissppshcsvkspvsspnn vtlrssvssp 301 aninnsrcsv sspsntnnrs tlsspaastv gsicspvnnafsytasgtsa gsstlrdvvp 361 spdtqekgaq evpfpkteev esaisngvtg qlnivqyikpepdgafsssc lggnskinsd 421 ssfsvpikqe stkhscsgts fkgnptvnpf pfmdgsyfsfmddkdyysls gilgppvpgf 481 dgncegsgfp vgikqepddg syypeasips saivgvnsggqsfhyrigaq gtislsrsar 541 dqsfqhlssf ppvntlvesw kshgdlssrr sdgypvleyipenvssstlr svstgssrps 601 kiclvcgdea sgchygvvtc gsckvffkra vegqhnylcagrndciidki rrkncpacrl 661 qkclqagmnl garkskklgk lkgiheeqpq qqqppppppppqspeegtty iapakepsvn 721 talvpqlsti sraltpspvm vleniepeiv yagydsskpdtaenllstln rlagkqmiqv 781 vkwakvlpgf knlpledqit liqyswmcls sfalswrsykhtnsqflyfa pdlvfneekm 841 hqsamyelcq gmhqislqfv rlqltfeeyt imkvllllstipkdglksqa afeemrtnyi 901 kelrkmvtkc pnnsgqswqr fyqltkllds mhdlvsdllefcfytfresh alkvefpaml 961 veiisdqlpk vesgnakply fhrk

Another non-limiting example of a ligand binding domain is a ligandbinding domain of a vitamin D receptor. For example, a ligand bindingdomain of a vitamin D receptor can be amino acids 124 to 426 of SEQ IDNO: 79. In some embodiments, a hormone receptor ligand binding domain isor includes a sequence that is at least 80%, at least 81%, at least 82%,at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identical to amino acids 124 to 426 of SEQ IDNO: 79.

(Human) SEQ ID NO: 79 1 meamaastsl pdpgdfdrnv pricgvcgdr atgfhfnamtcegckgffrr smkrkalftc 61 pfngdcritk dnrrhcqacr lkrcvdigmm kefiltdeevqrkremilkr keeealkdsl 121 rpklseeqqr iiailldahh ktydptysdf cqfrppvrvndgggshpsrp nsrhtpsfsg 181 dsssscsdhc itssdmmdss sfsnldlsee dsddpsvtlelsqlsmlphl adlvsysiqk 241 vigfakmipg frdltsedqi vllkssaiev imlrsnesftmddmswtcgn qdykyrvsdv 301 tkaghsleli eplikfqvgl kklnlheeeh vllmaicivspdrpgvqdaa lieaiqdrls 361 ntlqtyircr hpppgshlly akmiqkladl rslneehskqyrclsfqpec smkltplvle 421 vfgneis

Another non-limiting example of a ligand binding domain is a ligandbinding domain of a PPARα receptor. For example, a ligand binding domainof a PPARα receptor can be amino acids 201 to 467 of SEQ ID NO: 80. Insome embodiments, a hormone receptor ligand binding domain is orincludes a sequence that is at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identical to amino acids 201 to 467 of SEQ IDNO: 80.

(Human) SEQ ID NO: 80 1 mvdtesplcp lspleagdle splseeflqe mgniqeisqsigedssgsfg fteyqylgsc 61 pgsdgsvitd tlspasspss vtypvvpgsv despsgalniecricgdkas gyhygvhace 121 gckgffrrti rlklvydkcd rsckiqkknr nkcqycrfhkclsvgmshna irfgrmprse 181 kaklkaeilt cehdiedset adlkslakri yeaylknfnmnkvkarvils gkasnnppfv 241 ihdmetlcma ektlvaklva ngiqnkeaev rifhccqctsvetvteltef akaipgfanl 301 dlndqvtllk ygvyeaifam lssvmnkdgm lvaygngfitreflkslrkp fcdimepkfd 361 famkfnalel ddsdislfva aiiccgdrpg llnvghiekmqegivhvlrl hlqsnhpddi 421 flfpkllqkm adlrqlvteh aqlvqiikkt esdaalhpllqeiyrdmy

Another non-limiting example of a ligand binding domain is a ligandbinding domain of a PPARβ/δ receptor. For example, a ligand bindingdomain of a PPARβ/δ receptor can be amino acids 173 to 440 of SEQ ID NO:81. In some embodiments, a hormone receptor ligand binding domain is orincludes a sequence that is at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identical to amino acids 173 to 440 of SEQ IDNO: 81.

(Human) SEQ ID NO: 81 1 meqpqeeape vreeeekeev aeaegapeln ggpqhalpsssytdlsrsss ppslldqlqm 61 gcdgascgsl nmecrvcgdk asgfhygvha cegckgffrrtirmkleyek cersckiqkk 121 nrnkcqycrf qkclalgmsh nairfgrmpe aekrklvagltanegsqynp qvadlkafsk 181 hiynaylknf nmtkkkarsi ltgkashtap fvihdietlwqaekglvwkq lvnglppyke 241 isvhvfyrcq cttvetvrel tefaksipsf sslflndqvtllkygvheai famlasivnk 301 dgllvangsg fvtreflrsl rkpfsdiiep kfefavkfnalelddsdlal fiaaiilcgd 361 rpglmnvprv eaiqdtilra lefhlqanhp daqylfpkllqkmadlrqlv tehaqmmqri 421 kktetetslh pllqeiykdm y

Another non-limiting example of a ligand binding domain is a ligandbinding domain of a pregnane X receptor. For example, a ligand bindingdomain of a pregnane X receptor receptor can be amino acids 143 to 428of SEQ ID NO: 82. In some embodiments, a hormone receptor ligand bindingdomain is or includes a sequence that is at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% identical to amino acids 143 to428 of SEQ ID NO: 82.

(Human) SEQ ID NO: 82 1 mevrpkeswn hadfvhcedt esvpgkpsvn adeevggpqicrvcgdkatg yhfnvmtceg 61 ckgffrramk rnarlrcpfr kgaceitrkt rrqcqacrlrkclesgmkke mimsdeavee 121 rralikrkks ertgtqplgv qglteeqrmm irelmdaqmktfdttfshfk nfrlpgvlss 181 gcelpeslqa psreeaakws qvrkdlcslk vslqlrgedgsvwnykppad sggkeifsll 241 phmadmstym fkgiisfakv isyfrdlpie dqisllkgaafelcqlrfnt vfnaetgtwe 301 cgrlsycled taggfqqlll epmlkfhyml kklqlheeeyvlmqaislfs pdrpgvlqhr 361 vvdqlqeqfa itlksyiecn rpqpahrflf lkimamltelrsinaqhtqr llriqdihpf 421 atplmqelfg itgs

Another non-limiting example of a ligand binding domain is a ligandbinding domain of a liver X receptor. For example, a ligand bindingdomain of a liver X receptor can be amino acids 20 to 255 of SEQ ID NO:83. In some embodiments, a hormone receptor ligand binding domain is orincludes a sequence that is at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identical to amino acids 20 to 255 of SEQ IDNO: 83.

(Human) SEQ ID NO: 83 1 gshmsqgsge gegvqltaaq elmiqqlvaa qlqcnkrsfsdqpkvtpwpl gadpqsrdar 61 qqrfahftel aiisvqeivd fakqvpgflq lgredqiallkastieimll etarrynhet 121 ecitflkdft yskddfhrag lqvefinpif efsramrrlglddaeyalli ainifsadrp 181 nvqepgrvea lqqpyveall sytrikrpqd qlrfprmlmklvslrtlssv hseqvfalrl 241 qdkklpplls eiwdvhe

Another non-limiting example of a ligand binding domain is a ligandbinding domain of a farnesoid X receptor. For example, a ligand bindingdomain of a farnesoid X receptor can be amino acids 247 to 467 of SEQ IDNO: 84. In some embodiments, a hormone receptor ligand binding domain isor includes a sequence that is at least 80%, at least 81%, at least 82%,at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identical to amino acids 247 to 467 of SEQ IDNO: 84.

(Human) SEQ ID NO: 84 1 mgskmnlieh shlpttdefs fsenlfgvlt eqvagplgqnlevepysqys nvqfpqvqpq 61 issssyysnl gfypqqpeew yspgiyelrr mpaetlyqgetevaempvtk kprmgasagr 121 ikgdelcvvc gdrasgyhyn altcegckgf frrsitknavykcknggncv mdmymrrkcq 181 ecrlrkckem gmlaecllte iqckskrlrk nvkqhadqtvnedsegrdlr qvtsttkscr 241 ekteltpdqq tllhfimdsy nkqrmpqeit nkilkeefsaeenfliltem atnhvqvlve 301 ftkklpgfqt ldhedqiall kgsaveamfl rsaeifnkklpsghsdllee rirnsgisde 361 yitpmfsfyk sigelkmtqe eyalltaivi lspdrqyikdreaveklqep lldvlqklck 421 ihqpenpqhf acllgrltel rtfnhhhaem lmswrvndhkftpllceiwd vq

Another non-limiting example of a ligand binding domain is a ligandbinding domain of a RAR-related orphan receptor A. For example, a ligandbinding domain of a RAR-related orphan receptor A can be amino acids 217to 456 of SEQ ID NO: 85. In some embodiments, a hormone receptor ligandbinding domain is or includes a sequence that is at least 80%, at least81%, at least 82%, at least 83%, at least 84%, at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% identical to aminoacids 217 to 456 of SEQ ID NO: 85.

(Human) SEQ ID NO: 85 1 mmyfviaamk aqieiipcki cgdkssgihy gvitcegckgffrrsqqsna tyscprqknc 61 lidrtsrnrc qhcrlqkcla vgmsrdavkf grmskkqrdslyaevqkhrm qqqqrdhqqq 121 pgeaepltpt ynisanglte lhddlsnyid ghtpegskadsavssfyldi qpspdqsgld 181 ingikpepic dytpasgffp ycsftngets ptvsmaelehlaqniskshl etcqylreel 241 qqitwqtflq eeienyqnkq revmwqlcai kiteaiqyvvefakridgfm elcqndqivl 301 lkagslevvf irmcrafdsq nntvyfdgky aspdvfkslgcedfisfvfe fgkslcsmhl 361 tedeialfsa fvlmsadrsw lqekvkiekl qqkiqlalqhvlqknhredg iltklickvs 421 tlralcgrht eklmafkaiy pdivrlhfpp lykelftsefepamqidg

Another non-limiting example of a ligand binding domain is a ligandbinding domain of a RAR-related orphan receptor C. For example, a ligandbinding domain of a RAR-related orphan receptor C can be amino acids 268to 506 of SEQ ID NO: 86. In some embodiments, a hormone receptor ligandbinding domain is or includes a sequence that is at least 80%, at least81%, at least 82%, at least 83%, at least 84%, at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% identical to aminoacids 268 to 506 of SEQ ID NO: 86.

(Human) SEQ ID NO: 86 1 mdrapqrqhr asrellaakk thtsqievip ckicgdkssgihygvitceg ckgffrrsqr 61 cnaaysctrq qncpidrtsr nrcqhcrlqk clalgmsrdavkfgrmskkq rdslhaevqk 121 qlqqrqqqqq epvvktppag aqgadtltyt lglpdgqlplgsspdlpeas acppgllkas 181 gsgpsysnnl akaglngasc hleyspergk aegresfystgsqltpdrcg lrfeehrhpg 241 lgelgqgpds ygspsfrstp eapyasltei ehlvqsvcksyretcqlrle dllrqrsnif 301 sreevtgyqr ksmwemwerc ahhlteaiqy vvefakrlsgfmelcqndqi vllkagamev 361 vlvrmcrayn adnrtvffeg kyggmelfra lgcselissifdfshslsal hfsedeialy 421 talvlinahr pglqekrkve qlqynlelaf hhhlckthrqsilaklppkg klrslcsqhv 481 erlqifqhlh pivvqaafpp lykelfstet espvglsk

Another non-limiting example of a ligand binding domain is a ligandbinding domain of a retinoic acid receptor. For example, a ligandbinding domain of retinoic acid receptor can be amino acids 156 to 385of SEQ ID NO: 87. In some embodiments, a hormone receptor ligand bindingdomain is or includes a sequence that is at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% identical to amino acids 156 to385 of SEQ ID NO: 87.

(Human) SEQ ID NO: 87 1 mlgglsppga lttlqhqlpv sgystpspat ietqsssseeivpsppsppp lpriykpcfv 61 cqdkssgyhy gvsacegvkg ffrrsiqknm vytvhrdknciinkvtrnrc qycrlqkcfe 121 vgmskesvrn drnkkkkevp kpecsesytv tpevgeliekvrkahqetfp alcqlgkytt 181 nnsseqrvsl didlwdkfse lstkciiktv efakqlpgfttltiadqitl lkaacldili 241 lrictrytpe qdtmtfsdgl tlnrtqmhna gfgpltdlvfafanqllple mddaetglls 301 aiclicgdrq dleqpdrvdm lqepllealk vyvrkrrpsrhmfpkmlmki tdlrsisakg 361 aervitlkme ipgsmppliq emlensegld tlsgqpggggrdggglappp gscspslsps 421 snrsspaths p

As can be appreciated in the art, amino acids that are not conservedbetween different mammalian homologues of hormone receptor ligandbinding domains are more likely to not play an important role in thehormone or hormone analogue binding activity of the ligand bindingdomain, while those amino acids that are conserved between differentmammalian homologues of hormone receptor ligand binding domains are morelikely to play an important role in the hormone or hormone analoguebinding activity of the ligand binding domain. Some ligand bindingdomains can include one or more (e.g., two, three, four, five, or six)amino acid substitutions of amino acids that are not conserved betweendifferent mammalian homologues of hormone receptor ligand bindingdomains.

Antigen-Binding Domains

In some embodiments of the single-chain chimeric antigen receptors orthe multi-chain chimeric antigen receptors, the antigen-binding domaincan be selected from a scFv, a scFv-Fc, a VHH domain, a VNAR domain, a(scFv)₂, and a BiTE. Additional examples of antigen-binding domains thatcan be used in a single-chain chimeric antigen receptor or a multi-chainchimeric antigen receptor are known in the art.

A single-chain Fv or scFv fragment includes a V_(H) domain and a V_(L)domain in a single polypeptide chain. The V_(H) and V_(L) are generallylinked by a peptide linker. In other examples, the linker can be asingle amino acid. In some examples, the linker can be a chemical bond.See, e.g., Pluckthun, Antibodies from E. coli. In Rosenberg M. & MooreG. P. (Eds.), The Pharmacology of Monoclonal Antibodies, Vol. 113, pp.269-315, Spinger-Verlag, New York, 1994. An exemplary V_(L) and V_(H)sequences that can be used in a scFv are shown in SEQ ID NOs: 104 and108 in the Examples. An exemplary linker that can be used between theV_(L) and V_(H) domains in an scFv can be any of the exemplary linkersdescribed herein (e.g., a linker having the sequence of SEQ ID NO: 106)

Sc-Fv-Fc fragments include an scFv attached to an Fc domain. Forexample, an Fc domain can be attached, e.g., to the C-terminus of thescFv. The Fc domain can follow the V_(L) or V_(H), depending on theorientation of the variable domains in the scFv. The Fc domain can beany Fc domain known in the art. In some examples, the Fc domain is anIgG1, IgG2, IgG3, or IgG4 Fc domain (e.g., a human IgG1, IgG2, IgG3, orIgG4 Fc domain).

BiTEs are antigen-binding domains that include two V_(L) and two V_(H)in a single polypeptide that together form two scFvs, which can eachbind to different epitopes on the same antigen or each bind to differentantigens. See, e.g., Baeuerle et al., Curr. Opin. Mol. Ther. 11:22-30,2009; Wolf et al., Drug Discovery Today 10:1237-1244, 2005; and Huehlset al., Immunol. Cell Biol. 93:290-296, 2015.

A V_(H)H domain is a single monomeric variable antibody domain found incamelids, and a V_(NAR) domain is a single monomeric variable antibodydomain found in cartilaginous fish. V_(H)H domains and V_(NAR) domainsare described in, e.g., Van Audenhove et al., EBioMedicine 8:40-48,2016; Krah et al., Immunopharmacol. Immunotoxicol. 38:21-28, 2016;Cromie et al., Curr. Top. Med. Chem. 15:2543-2557, 2016; Kijanka et al.,Nanomedicine 10:161-174, 2015; Kovaleva et al., Expert. Opin. Biol.Ther. 14:1527-1539, 2014; De Meyer et al., Trends Biotechnol.32:263-270, 2014; Mujic-Delic et al., Trends Pharmacol. Sci. 35:247-255,2014; Muyldermans, Ann. Rev. Biochem. 82:775-797, 2013; Vincke et al.,Methods Mol. Biol. 911:15-26, 2012; Rahbarizadeh et al., Immunol.Invest. 40:299-338, 2011; Van Bockstaele et al., Curr. Opin. Investig.Drugs 10:1212-1224, 2009; Wesolowski et al., Med. Microbiol. Immunol.198:157-174, 2009; De Genst et al., Dev. Comp. Immunol. 30:187-198,2006; Muyldermans, J. Biotechnol. 74:277-302, 2001; and Muyldermans etal., Trends Biochem. Sci. 26:230-235, 2001.

In some embodiments of the multi-chain chimeric antigen receptors, atleast two of the polypeptides that make up the multi-chain chimericantigen receptor can interact together to form an antigen-bindingdomain. In such embodiments, the antigen-binding domain can be selectedfrom the group of an antigen-binding antibody fragment, a dual-affinityre-targeting antibody (DART), Fab-scFv-Fc, a triomab, a crossmab, anortho-Fab IgG, IgG-scFv, scFv₂-Fc, a bi-nanobody, tanden antibody, aDART-Fc, a scFv-HAS-scFv, DNL-Fab₃, DAF (two-in-one or four-in-one),DutaMab, DT-IgG knobs-in-holes common LC, knobs-in-holes assembly,Fab-arm exchange antibody, SEEDbody, Triomab, LUZ-Y, scDiabody-Fc, Fcab,kλ-body, orthogonal Fab, DVD-IgG IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv,scFv-(L)-IgG IgG (L,H)-Fc, IgG(H)-V, V(H)-IgG IgG(L)-V, V(L)-IgG KIHIgG-scFab, 2scFv-IgG IgG-2scFv, scFv4-Ig, Zybody, DVI-IgG, nanobody,nanobody-HSA, a diabody, a TandAb, scDiabody, scDiabody-CH3, charge pairantibody, diabody-CH3, Cov-X-Bod, Triple Body, miniantibody, a DVD-Ig,minibody, a 2-in-1-IgG TriBi minibody, scFv-CH3 KIH, Fab-scFv,scFv-CH-CL-scFv, F(ab′)₂-scFV₂, scFv-KIH, tetravalent HCAb, diabody-Fc,tandem scFv-Fc, intrabody, dock and lock bispecific antibody, ImmTAC,HSAbody, scDiabody-HAS, tandem scFv, IgG-IgG and scFv1-PEG-scFv₂.

Non-limiting examples of an antigen-binding antibody fragments includean Fv fragment, a Fab fragment, a F(ab′)₂ fragment, and a Fab′ fragment.Additional examples of an antigen-binding antibody fragment is anantigen-binding fragment of an IgG (e.g., an antigen-binding fragment ofIgG1, IgG2, IgG3, or IgG4) (e.g., an antigen-binding fragment of a humanor humanized IgG e.g., human or humanized IgG1, IgG2, IgG3, or IgG4); anantigen-binding fragment of an IgA (e.g., an antigen-binding fragment ofIgA1 or IgA2) (e.g., an antigen-binding fragment of a human or humanizedIgA, e.g., a human or humanized IgA1 or IgA2); an antigen-bindingfragment of an IgD (e.g., an antigen-binding fragment of a human orhumanized IgD); an antigen-binding fragment of an IgE (e.g., anantigen-binding fragment of a human or humanized IgE); or anantigen-binding fragment of an IgM (e.g., an antigen-binding fragment ofa human or humanized IgM).

Examples of DVD-Igs are described, e.g., in DiGiammarino et al., MethodsMol. Biol. 899:145-156, 2012; Jakob et al., MABs 5:358-363, 2013; andU.S. Pat. Nos. 7,612,181; 8,258,268; 8,586,714; 8,716,450; 8,722,855;8,735,546; and 8,822,645. Examples of DARTs are described in, e.g.,Garber, Nature Reviews Drug Discovery 13:799-801, 2014. Examples oftriomabs, kih IgG with a common LCs, crossmabs, ortho-Fab IgGs,2-in-1-IgGs, IgG-ScFvs, scFv2-Fcs, bi-nanobodies, tanden antibodies,DART-Fcs, scFv-HAS-scFvs, and DNL-Fab3s are described in, e.g.,Kontermann et al., Drug Discovery Today 20:838-847, 2015. Examples ofDAFs (two-in-one or four-in-one), DutaMabs, DT-IgGs, knobs-in-holescommon LCs, knobs-in-holes assemblies, charge pair antibodies, Fab-armexchange antibodies, SEEDbodies, Triomabs, LUZ-Ys, Fcabs, kλ-bodies,orthogonal Fabs, DVD-IgGs, IgG(H)-scFvs, scFv-(H)IgGs, IgG(L)-scFvs,scFv-(L)-IgGs, IgG (L,H)-Fcs, IgG(H)-Vs, V(H)-IgGs, IgG(L)-Vs,V(L)-IgGs, KIH IgG-scFabs, 2scFv-IgGs, IgG-2scFvs, scFv4-Igs, Zybodies,DVI-IgGs, nanobodies, nanobody-HSAs, diabodies, TandAbs, scDiabodies,scDiabody-CH3s, Diabody-CH3s, Triple Bodies, miniantibodies, minibodies,TriBi minibodies, scFv-CH3 KIHs, Fab-scFvs, scFv-CH-CL-scFvs,F(ab′)₂-scFV2s, scFv-KIHs, Fab-scFv-Fcs, tetravalent HCAbs,scDiabody-Fcs, diabody-Fcs, tandem scFv-Fcs, intrabodies, dock and lockbispecific antibodies, ImmTACs, HSAbodies, scDiabody-HASs, tandem scFvs,IgG-IgGs, Cov-X-Bodies, and scFv1-PEG-scFv2s are described in, e.g.,Spiess et al., Mol. Immunol. 67:95-106, 2015.

Any of the antigen-binding domains described herein can bind to anantigen with a dissociation equilibrium constant (K_(D)) of less than1×10⁻⁷ M, less than 1×10⁻⁸ M, less than 1×10⁻⁹ M, less than 1×10⁻¹⁰ M,less than 1×10⁻¹¹ M, less than 1×10⁻¹² M, or less than 1×10⁻¹³ M. Insome embodiments, the antigen-binding protein complexes provided hereincan bind to a first and/or second antigen with a K_(D) of about 1×10⁻⁴ Mto about 1×10⁻⁶ M, about 1×10⁻⁵ M to about 1×10⁻⁷ M, about 1×10⁻⁶ M toabout 1×10⁻⁸ M, about 1×10⁻⁷ M to about 1×10⁻⁹ M, about 1×10⁻⁸ M toabout 1×10⁻¹⁰ M, or about 1×10⁻⁹ M to about 1×10⁻¹¹ M (inclusive). Avariety of different methods known in the art can be used to determinethe K_(D) value of an antigen-binding domain (e.g., an electrophoreticmobility shift assay, a filter binding assay, surface plasmon resonance,and a biomolecular binding kinetics assay, etc.).

Antigens

In some embodiments, an antigen-binding domain described herein can bindto a single antigen (e.g., any of the exemplary antigens describedherein or known in the art). In some embodiments, an antigen-bindingdomain described herein can bind to two or more different antigens(e.g., two or more of any of the exemplary antigens described herein orknown in the art). Non-limiting examples of antigens include: HER2, A33antigen, 9-0-acetyl-GD3, CA19-9 marker, BhCG, CA-125 marker,carboanhydrase IX (MN/CA IX), calreticulin, CCR5, CCR8, CD2, CD3, CD5,CD16, CD19, CD20, CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD40L,CD44, CD44V6, CD63, CD70, CD84, CD96, CD100, CC123, CD133, CD137, CD138,CD150, CD152 (CTLA-4), CD160, CRTAM, CS1 (CD319), DNAM-1 (CD226), CD229,CD244, CD272 (BTLA), CD274 (PDL-1, B7H1), CD279 (PD-1), CD319, CD352,CRTAM (CD355), CD358, DR3, GITR (TNFRSF 18), HVEM, ICOS, LIGHT, LTBR,OX40, activating forms of KIR, NKG2C, NKG2D, NKG2E, one or more naturalcytotoxicity receptors, NTB-A, PEN-5, carcinoma embryonic antigen (CEA;CD66e), desmoglein 4, E-cadherin neoepitope, endosialin, ephrin A2(EphA2), epidermal growth factor receptor (EGFR), epithelial celladhesion molecule (EpCAM), fucosyl GM1, GD2, GD3, GM2, ganglioside GM3,Globo H, glycoprotein 100, HER2/neu, HER3, HER4, insulin-like growthfactor receptor 1, Lewis-Y, LG, Ly-6, melanoma-specificchondroitin-sulfate proteoglycan (MCSCP), mesothelin, MUC1, MUC2, MUC3,MUC4, MUC5AC, MUC5_(b), MUC7, MUC16, Mullerian inhibitory substance(MIS) receptor type II, plasma cell antigen, poly SA, PSCA, PSMA, sonichedgehog (SHH), SAS, STEAP, sTn antigen, TNF-α precursor, 2B4 (CD244),β2-integrins, KIR, KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL2, KIR-L, KLRGI,LAIR-1, NKG2A, NKR-P IA, Siglec-3, Siglec-7, Siglec-9, TCRa, TCRB,TCR5y, TIM1, LAG3, LAIR1, PD-1H, TIGIT, TIM2, and TIM3. Additionalexamples of antigens are known in the art.

Costimulatory Domains

In normal lymphocytes, T cell activation is mediated by two classes ofintracellular signaling domains. Primary signaling is initiated viaMHC-mediated antigen-dependent activation via the T cell receptor (e.g.,a TCR/CD3 complex). A secondary or costimulatory signal is provided by adifferent receptor that includes a costimulatory signaling domain, whichacts in an antigen-independent manner. Signals generated through thesignaling domain of the TCR alone are insufficient for complete T cellactivation; a co-stimulatory signal is also required.

In some embodiments, a single-chain chimeric polypeptide, a single-chainchimeric antigen receptor, or a multi-chain chimeric antigen receptorincludes a costimulatory domain, or portion thereof, from an endogenousmammalian (e.g., human) transmembrane polypeptide selected from thegroup of: CD27 (also known as S152, S152.LPFS2, T14, TNFRSF7, and Tp55),CD28 (also known as Tp44), 4-1BB (also known as TNFRSF9, CD137, CDw137,ILA, and tumor necrosis factor receptor superfamily member 9), OX40(also known as TNFRSF4, ACT35, RP5-902P8.3, IMD16, CD134, TXGP1L, andtumor necrosis factor receptor superfamily member 4), CD30 (also knownas TNFRSF8, D1S166E, and Ki-1), CD40L (also known as CD40LG, CD154,HIGM1, IGM, IMD3, T-BAM, TNFSF5, TRAP, gp39, hCD40L, and CD40 ligand),CD40 (also known as Bp50, CDW40, TNFRSF5, p50, CD40 (protein), and CD40molecule), PD-1 (also known as PDCD1, CD279, PD-1, SLEB2, hPD-1, hPD-1,hSLE1, and Programmed cell death 1), PD-L1 (also known as CD274, B7-H,B7H1, PD-L1, PDCD1L1, PDCD1LG1, PDL1, CD274 molecule, and Programmedcell death 1 ligand 1), ICOS (also known as AILIM, CD278, and CVID1),LFA-1 (also known as Lymphocyte function-associated antigen 1), CD2(also known as LFA-2, SRBC, T11, and CD2 molecule), CD7 (also known asGP40, LEU-9, TP41, Tp40, and CD7 molecule), CD160 (also known as BY55,NK1, NK28, and CD160 molecule), LIGHT (also known as TNFSF14, CD258,HVEML, LIGHT, LTg, TR2, TNLG1D, and tumor necrosis factor superfamilymember 14), BTLA (also known as CD272 and BTLA1), TIM3 (also known asHAVCR2, HAVcr-2, KIM-3, TIM3, TIMD-3, TIMD3, Tim-3, CD366, and hepatitisA virus cellular receptor 2), CD244 (also known as 2B4, NAIL, NKR2B4,Nmrk, SLAMF4, and CD244 molecule), CD80 (also known as B7, B7-1, B7.1,BB1, CD28LG, CD28LG1, LAB7, and CD80 molecule), LAG3 (also known asCD223 and lymphocyte activating 3), NKG2C (also known as CD314,D12S2489E, KLR, NKG2-D, NKG2D, and killer cell lectin like receptor K1),GITR (also known as TNFRSF18, RP5-902P8.2, AITR, CD357, and GITR-D),HVEM (also known as TNFRSF14, RP3-395M20.6, ATAR, CD270, HVEA, HVEM,LIGHTR, and TR2), TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9,TLRIO, CARD11, CD54 (ICAM), CD83, DAP10, LAT, SLP76, TRIM, ZAP70, andB7-H3 (also known as CD276, 4Ig-B7-H3, B7H3, B7RP-2, and CD276molecule). The letters “CD” is the previous sentence stand for “Clusterof Differentiation.” For example, CD3 stands for “Cluster ofDifferentiation 3.” In some embodiments, a single-chain chimericpolypeptide, a single-chain chimeric antigen receptor, or a multi-chainchimeric antigen receptor includes a costimulatory domain, or portionthereof, from an endogenous mammalian (e.g., human) transmembranepolypeptide (e.g., a mammalian or human homolog of any of thepolypeptides listed above).

Any costimulatory domain, or portion thereof, that serves to provide acostimulatory signal is suitable for use in accordance with compositionsand methods disclosed herein. In some embodiments, a single-chainchimeric polypeptide, a single-chain chimeric antigen receptor, or amulti-chain chimeric antigen receptor includes a costimulatory domain,or portion thereof, from human CD28 (e.g. Accession No. P01747, e.g.,from amino acids 180 to 220 of SEQ ID NO: 18). In some embodiments, acostimulatory domain is or includes a sequence that is at least 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical (or is identical) to amino acids180 to 220 of SEQ ID NO: 18, or a fragment thereof. In some embodiments,a costimulatory domain is or includes a sequence that is at least 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% (or is identical) to SEQ ID NO: 116, or afragment thereof.

SEQ ID NO: 18 MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPG PTRKHYQPYAPPRDFAAYRS

In some embodiments, a single-chain chimeric polypeptide, a single-chainchimeric antigen receptor, or a multi-chain chimeric antigen receptorincludes a costimulatory domain, or portion thereof, from human 4-1BB(e.g. Accession No. Q07011, e.g., from amino acids 214 to 255 of SEQ IDNO: 19). In some embodiments, a costimulatory domain is or includes asequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical toamino acids 214 to 255 of SEQ ID NO: 19, or a portion thereof.

SEQ ID NO: 19 MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE GGCEL

In some embodiments, a costimulatory domain is or includes a sequencethat is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:44, 57, or 116, or a portion thereof.

A single-chain chimeric polypeptide, a single-chain chimeric antigenreceptor, or a multi-chain chimeric antigen receptor disclosed hereincan include a costimulatory domain that includes a sequence of aminoacids from any isoform of an endogenous mammalian (e.g., human)transmembrane polypeptide having a costimulatory domain including, e.g.,an isoform of: CD27, CD28, 4-1BB, OX40, CD30, CD40L, CD40, PD-1, PD-L1,ICOS, LFA-1, CD2, CD7, CD160, LIGHT, BTLA, TIM3, CD244, CD80, LAG3,NKG2C, or B7-H3 (including, without limitation, a mammalian or humanhomolog of any of these polypeptides).

In some embodiments, a costimulatory domain, or portion thereof, of asingle-chain chimeric polypeptide, a single-chain chimeric antigenreceptor, or a multi-chain chimeric antigen receptor includes a sequenceof amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to acostimulatory domain from one or more of a mammalian (e.g., human) CD27,CD28, 4-1BB, OX40, CD30, CD40L, CD40, PD-1, PD-L1, ICOS, LFA-1, CD2,CD7, CD160, LIGHT, BTLA, TIM3, CD244, CD80, LAG3, NKG2C, or B7-H3. Insome embodiments, a costimulatory domain, or portion thereof, of asingle-chain chimeric polypeptide, a single-chain chimeric antigenreceptor, or a multi-chain chimeric antigen receptor includes a sequenceof amino acids having one or more amino acid substitutions, deletions,or additions as compared to a costimulatory domain of one or more of anendogenous mammalian (e.g., human) transmembrane polypeptide: an α chainof a T cell receptor, a β chain of the T cell receptor, a ζ chain of theT cell receptor, CD27, CD28, 4-1BB, OX40, CD30, CD40L, CD40, PD-1,PD-L1, ICOS, LFA-1, CD2, CD7, CD160, LIGHT, BTLA, TIM3, CD244, CD80,LAG3, NKG2C, or B7-H3 (including, without limitation, a mammalian orhuman homolog of any of these polypeptides).

In some embodiments, a single-chain chimeric polypeptide, a single-chainchimeric antigen receptor, or a multi-chain chimeric antigen receptorincludes a costimulatory domain that is a chimeric costimulatory domainhaving portions of a costimulatory domain from two or more endogenousmammalian (e.g., human) transmembrane polypeptides including, withoutlimitation, CD27, CD28, 4-1BB, OX40, CD30, CD40L, CD40, PD-1, PD-L1,ICOS, LFA-1, CD2, CD7, CD160, LIGHT, BTLA, TIM3, CD244, CD80, LAG3,NKG2C, or B7-H3 (including, without limitation, a mammalian or humanhomolog of any of these polypeptides), such that the two or moreportions of the transmembrane domains together constitute a functionalcostimulatory domain. In some embodiments, such a portion of a chimericcostimulatory domain can include one or more amino acid substitutions,deletions, or additions as compared to a corresponding portion of awildtype costimulatory domain.

A costimulatory domain of a single-chain chimeric polypeptide, asingle-chain chimeric antigen receptor, or a multi-chain chimericantigen receptor disclosed herein can be of any suitable length. Forexample, a costimulatory domain can have a length of about 20 aminoacids to about 200 amino acids, about 190 amino acids, about 180 aminoacids, about 170 amino acids, about 160 amino acids, about 150 aminoacids, about 140 amino acids, about 130 amino acids, about 120 aminoacids, about 110 amino acids, about 100 amino acids, about 95 aminoacids, about 90 amino acids, about 85 amino acids, about 80 amino acids,about 75 amino acids, about 70 amino acids, about 65 amino acids, about60 amino acids, about 55 amino acids, about 50 amino acids, about 45amino acids, about 40 amino acids, about 35 amino acids, about 30 aminoacids, or about 25 amino acids (inclusive); about 25 amino acids toabout 200 amino acids, about 190 amino acids, about 180 amino acids,about 170 amino acids, about 160 amino acids, about 150 amino acids,about 140 amino acids, about 130 amino acids, about 120 amino acids,about 110 amino acids, about 100 amino acids, about 95 amino acids,about 90 amino acids, about 85 amino acids, about 80 amino acids, about75 amino acids, about 70 amino acids, about 65 amino acids, about 60amino acids, about 55 amino acids, about 50 amino acids, about 45 aminoacids, about 40 amino acids, about 35 amino acids, or about 30 aminoacids (inclusive); about 30 amino acids to about 200 amino acids, about190 amino acids, about 180 amino acids, about 170 amino acids, about 160amino acids, about 150 amino acids, about 140 amino acids, about 130amino acids, about 120 amino acids, about 110 amino acids, about 100amino acids, about 95 amino acids, about 90 amino acids, about 85 aminoacids, about 80 amino acids, about 75 amino acids, about 70 amino acids,about 65 amino acids, about 60 amino acids, about 55 amino acids, about50 amino acids, about 45 amino acids, about 40 amino acids, or about 35amino acids (inclusive); about 35 amino acids to about 200 amino acids,about 190 amino acids, about 180 amino acids, about 170 amino acids,about 160 amino acids, about 150 amino acids, about 140 amino acids,about 130 amino acids, about 120 amino acids, about 110 amino acids,about 100 amino acids, about 95 amino acids, about 90 amino acids, about85 amino acids, about 80 amino acids, about 75 amino acids, about 70amino acids, about 65 amino acids, about 60 amino acids, about 55 aminoacids, about 50 amino acids, about 45 amino acids, or about 40 aminoacids (inclusive); about 40 amino acids to about 200 amino acids, about190 amino acids, about 180 amino acids, about 170 amino acids, about 160amino acids, about 150 amino acids, about 140 amino acids, about 130amino acids, about 120 amino acids, about 110 amino acids, about 100amino acids, about 95 amino acids, about 90 amino acids, about 85 aminoacids, about 80 amino acids, about 75 amino acids, about 70 amino acids,about 65 amino acids, about 60 amino acids, about 55 amino acids, about50 amino acids, or about 45 amino acids (inclusive); about 45 aminoacids to about 200 amino acids, about 190 amino acids, about 180 aminoacids, about 170 amino acids, about 160 amino acids, about 150 aminoacids, about 140 amino acids, about 130 amino acids, about 120 aminoacids, about 110 amino acids, about 100 amino acids, about 95 aminoacids, about 90 amino acids, about 85 amino acids, about 80 amino acids,about 75 amino acids, about 70 amino acids, about 65 amino acids, about60 amino acids, about 55 amino acids, or about 50 amino acids(inclusive); about 50 amino acids to about 200 amino acids, about 190amino acids, about 180 amino acids, about 170 amino acids, about 160amino acids, about 150 amino acids, about 140 amino acids, about 130amino acids, about 120 amino acids, about 110 amino acids, about 100amino acids, about 95 amino acids, about 90 amino acids, about 85 aminoacids, about 80 amino acids, about 75 amino acids, about 70 amino acids,about 65 amino acids, about 60 amino acids, or about 55 amino acids(inclusive); about 55 amino acids to about 200 amino acids, about 190amino acids, about 180 amino acids, about 170 amino acids, about 160amino acids, about 150 amino acids, about 140 amino acids, about 130amino acids, about 120 amino acids, about 110 amino acids, about 100amino acids, about 95 amino acids, about 90 amino acids, about 85 aminoacids, about 80 amino acids, about 75 amino acids, about 70 amino acids,about 65 amino acids, or about 60 amino acids (inclusive); about 60amino acids to about 200 amino acids, about 190 amino acids, about 180amino acids, about 170 amino acids, about 160 amino acids, about 150amino acids, about 140 amino acids, about 130 amino acids, about 120amino acids, about 110 amino acids, about 100 amino acids, about 95amino acids, about 90 amino acids, about 85 amino acids, about 80 aminoacids, about 75 amino acids, about 70 amino acids, or about 65 aminoacids (inclusive); about 65 amino acids to about 200 amino acids, about190 amino acids, about 180 amino acids, about 170 amino acids, about 160amino acids, about 150 amino acids, about 140 amino acids, about 130amino acids, about 120 amino acids, about 110 amino acids, about 100amino acids, about 95 amino acids, about 90 amino acids, about 85 aminoacids, about 80 amino acids, about 75 amino acids, or about 70 aminoacids (inclusive); about 70 amino acids to about 200 amino acids, about190 amino acids, about 180 amino acids, about 170 amino acids, about 160amino acids, about 150 amino acids, about 140 amino acids, about 130amino acids, about 120 amino acids, about 110 amino acids, about 100amino acids, about 95 amino acids, about 90 amino acids, about 85 aminoacids, or about 80 amino acids (inclusive); about 80 amino acids toabout 200 amino acids, about 190 amino acids, about 180 amino acids,about 170 amino acids, about 160 amino acids, about 150 amino acids,about 140 amino acids, about 130 amino acids, about 120 amino acids,about 110 amino acids, about 100 amino acids, about 95 amino acids, orabout 90 amino acids (inclusive); about 90 amino acids to about 200amino acids, about 190 amino acids, about 180 amino acids, about 170amino acids, about 160 amino acids, about 150 amino acids, about 140amino acids, about 130 amino acids, about 120 amino acids, about 110amino acids, or about 100 amino acids (inclusive); about 100 amino acidsto about 200 amino acids, about 190 amino acids, about 180 amino acids,about 170 amino acids, about 160 amino acids, about 150 amino acids,about 140 amino acids, about 130 amino acids, about 120 amino acids, orabout 110 amino acids (inclusive); about 110 amino acids to about 200amino acids, about 190 amino acids, about 180 amino acids, about 170amino acids, about 160 amino acids, about 150 amino acids, about 140amino acids, about 130 amino acids, or about 120 amino acids(inclusive); about 120 amino acids to about 200 amino acids, about 190amino acids, about 180 amino acids, about 170 amino acids, about 160amino acids, about 150 amino acids, about 140 amino acids, or about 130amino acids (inclusive); about 130 amino acids to about 200 amino acids,about 190 amino acids, about 180 amino acids, about 170 amino acids,about 160 amino acids, about 150 amino acids, or about 140 amino acids(inclusive); about 140 amino acids to about 200 amino acids, about 190amino acids, about 180 amino acids, about 170 amino acids, about 160amino acids, or about 150 amino acids (inclusive); about 150 amino acidsto about 200 amino acids, about 190 amino acids, about 180 amino acids,about 170 amino acids, or about 160 amino acids (inclusive); about 160amino acids to about 200 amino acids, about 190 amino acids, about 180amino acids, or about 170 amino acids (inclusive); about 170 amino acidsto about 200 amino acids, about 190 amino acids, or about 180 aminoacids (inclusive); about 180 amino acids to about 200 amino acids orabout 190 amino acids (inclusive); or about 190 amino acids to about 200amino acids (inclusive).

In some embodiments, a single-chain chimeric polypeptide, a single-chainchimeric antigen receptor, or a multi-chain chimeric antigen receptorincludes two or more costimulatory domains, e.g., two, three, four, orfive, or more costimulatory domains. In some embodiments, the two ormore costimulatory domains are identical (e.g., they have the same aminoacid sequence). In some embodiments, the costimulatory domains are notidentical. For example, the costimulatory domains can be selected fromdifferent endogenous mammalian (e.g., human) transmembrane polypeptidesincluding, without limitation, CD27, CD28, 4-1BB, OX40, CD30, CD40L,CD40, PD-1, PD-L1, ICOS, LFA-1, CD2, CD7, CD160, LIGHT, BTLA, TIM3,CD244, CD80, LAG3, NKG2C, or B7-H3 (including, without limitation, amammalian or human homolog of any of these polypeptides). In someembodiments, the two or more costimulatory domains can differ from eachother by one or more (e.g., two, three, four, or five) amino acidsubstitutions, deletions, or additions. In some embodiments, the two ormore costimulatory domains exhibit at least 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity to each other.

Immunoreceptor Tyrosine-Based Activation Motifs (ITAMs)

ITAMs include a tyrosine separated from a leucine or isoleucine by anytwo other amino acids, and can thus be represented as, e.g.,Tyr-X-X-Leu/Ile. ITAMs are typically repeated (e.g., two or more times)in the cytoplasmic tails of certain cell surface proteins of the immunesystem, and are typically separated by between six and eight aminoacids.

In some embodiments, a single-chain chimeric polypeptide, a single-chainchimeric antigen receptor, or a multi-chain chimeric antigen receptorincludes an ITAM, or portion thereof, from an endogenous mammalian(e.g., human) polypeptide, wherein endogenous mammalian (e.g., human)polypeptide is selected from the group of: CD3ζ (also referred to as CD3zeta), CD3Δ (CD3 delta), CD3ε (CD3 epsilon), CD3γ (CD3 gamma), DAP12,FCεR1γ (Fc epsilon receptor I gamma chain), FcRy, FcRft, CD35, CD22,CD79A (antigen receptor complex-associated protein alpha chain), CD79B(antigen receptor complex-associated protein beta chain), and CD66d. Theletters “CD” is the previous sentence stand for “Cluster ofDifferentiation.” For example, CD3 stands for “Cluster ofDifferentiation 3.”

Any ITAM, or portion thereof, that serves to mediate signaling in anendogenous mammalian (e.g., human) transmembrane protein suitable foruse in accordance with compositions and methods disclosed herein. Insome embodiments, a single-chain chimeric polypeptide, a single-chainchimeric antigen receptor, or a multi-chain chimeric antigen receptorincludes an ITAM, or portion thereof, from human CD3 zeta (e.g.Accession No. P20963, e.g., an ITAM present in amino acids 52-164 of SEQID NO: 17, or a portion thereof; or SEQ ID NO: 118 or a portionthereof). In some embodiments, an ITAM comprises a sequence that is atleast 80% (e.g., at least 82%, at least 84%, at least 86%, at least 88%,at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, atleast 99%, or 100% identical to: the sequence of amino acids 52-165 ofSEQ ID NO: 17 (or a portion thereof), or the sequence of SEQ ID NO: 118(or a portion thereof).

SEQ ID NO: 17 MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK DTYDALHMQALPPR

In some embodiments, an ITAM comprises a sequence that is or includes asequence that is at least 80% (e.g., at least 82%, at least 84%, atleast 86%, at least 88%, at least 90%, at least 92%, at least 94%, atleast 96%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:48, 61, or 118, or a portion thereof.

As will be appreciated by those of ordinary skill in the art, certainpolypeptides have two or more isoforms that differ at least in theirprimary polypeptide sequence. For example, different isoforms can begenerated as a result of alternative splicing. A single-chain chimericpolypeptide, a single-chain chimeric antigen receptor, or a multi-chainchimeric antigen receptor disclosed herein can include an ITAM thatincludes a sequence of amino acids from any isoform of an endogenousmammalian transmembrane polypeptide having an ITAM including, e.g., amammalian (e.g., human) isoform of: CD3ζ, CD3D, CD3E, CD3G, DAP12,FCER1G, FcRy, FcRft, CD35, CD22, CD79A, CD79B, or CD66d.

In some embodiments, an ITAM, or portion thereof, of a single-chainchimeric polypeptide, a single-chain chimeric antigen receptor, or amulti-chain chimeric antigen receptor includes a sequence of amino acidshaving one or more (e.g., two, three, four, or five) amino acidsubstitutions, deletions, or additions as compared to an ITAM of one ormore of an ITAM in an endogenous mammalian (e.g., human) transmembraneprotein, such as, CD3ζ, CD3D, CD3E, CD3G, DAP12, FCER1G, FcRy, FcRft,CD35, CD22, CD79A, CD79B, or CD66d. For example, the tyrosine andleucine or isoleucine of an ITAM could be retained, while the two aminoacids separating them could be replaced with different amino acids.

In some embodiments, a single-chain chimeric polypeptide, a single-chainchimeric antigen receptor, or a multi-chain chimeric antigen receptorincludes an ITAM that is a chimeric ITAM having portions of an ITAM fromtwo or more endogenous mammalian (e.g., human) transmembranepolypeptides including, without limitation, CD3ζ, CD3D, CD3E, CD3G,DAP12, FCER1G, FcRy, FcRft, CD35, CD22, CD79A, CD79B, or CD66d(including, without limitation, a mammalian or human homolog of any ofthese polypeptides), such that the two or more ITAM portions togetherconstitute a functional ITAM. In some embodiments, such a portion of achimeric ITAM can include one or more amino acid substitutions,deletions, or additions as compared to a corresponding portion of a wildtype ITAM.

In some embodiments, a single-chain chimeric polypeptide, a single-chainchimeric antigen receptor, or a multi-chain chimeric antigen receptorincludes two or more ITAMs, e.g., two, three, four, or five, or moreITAMs. In some embodiments, the two or more ITAMs are identical (e.g.,they have the same amino acid sequence). In some embodiments, the two ormore ITAMs are not identical. For example, the ITAMs can be selectedfrom different endogenous mammalian (e.g., human) transmembranepolypeptides including, without limitation, CD3ζ, CD3D, CD3E, CD3G,DAP12, FCER1G, FcRy, FcRft, CD35, CD22, CD79A, CD79B (including, withoutlimitation, a mammalian or human homolog of any of these polypeptides).In some embodiments, the two or more ITAMs can differ from each other byone or more amino acid substitutions, deletions, or additions.

Nucleic Acids

Also provided herein are nucleic acids that encode any of thesingle-chain chimeric polypeptides, single-chain chimeric antigenreceptors, and multi-chain chimeric antigen receptors. Also providedherein are a set of nucleic acids that encode one or more single-chainpolypeptides that make up a multi-chain chimeric polypeptide (e.g.,where at least one single-chain polypeptide that makes up themulti-chain chimeric polypeptide is encoded by each nucleic acid in theset).

Vectors

Provided herein are vectors that include any of the nucleic acidsprovided herein. A “vector” according to the present disclosure is apolynucleotide capable of inducing the expression of a recombinantprotein (e.g., a single-chain chimeric polypeptide, a single-chainchimeric antigen receptor, or a multi-chain chimeric antigen receptor)in a mammalian cell. A vector provided herein can be, e.g., in circularor linearized form. Non-limiting examples of vectors include plasmids,SV40 vectors, adenoviral viral vectors, and adeno-associated virus (AAV)vectors. Non-limiting examples of vectors include lentiviral vectors orretroviral vectors, e.g., gamma-retroviral vectors. See, e.g., Carlenset al., Exp. Hematol. 28(10:1137-1146, 2000; Park et al., TrendsBiotechnol. 29(11):550-557, 2011; and Alonso-Camino et al., Mol. Ther.Nucleic Acids 2:e93, 2013. Non-limiting examples of retroviral vectorsinclude those derived from Moloney murine leukemia virus,myeloproliferative sarcoma virus, murine embryonic stem cell virus,murine stem cell virus, spleen focus forming virus, or adeno-associatedvirus. Non-limiting examples of retroviral vectors are described in,e.g., U.S. Pat. Nos. 5,219,740 and 6,207,453; Miller et al.,BioTechniques 7:980-990, 1989; Miller, Human Gene Therapy 1:5-14, 1990;Scarpa et al., Virology 180:849-852, 1991; Burns et al., Proc. Natl.Acad. Sci. U.S.A. 90:8033-8037, 1993; and Boris-Lawrie et al., Cur.Opin. Genet. Develop. 3:102-109, 1993. Exemplary lentiviral vectors aredescribed in, e.g., Wang et al., J. Immunother. 35(9):689-701, 2003;Cooper et al., Blood 101:1637-1644, 2003; Verhoeyen et al., Methods Mol.Biol. 506:97-114, 2009; and Cavalieri et al., Blood 102(2):497-505,2003.

Exemplary vectors, in which any of the nucleic acids provided herein canbe inserted, are described in, e.g., Ausubel et al., Eds. “CurrentProtocols in Molecular Biology” Current Protocols, 1993; and Sambrook etal., Eds. “Molecular Cloning: A Laboratory Manual,” 2nd ed., Cold SpringHarbor Press, 1989.

In some embodiments, the vectors further include a promoter and/orenhancer operably linked to any of the nucleic acids described herein.Non-limiting examples of promoters include promoters from humancytomegalovirus (CMV), mouse phosphoglycerate kinase 1, polyomaadenovirus, thyroid stimulating hormone α, vimentin, simian virus 40(SV40), tumor necrosis factor, β-globin, α-fetoprotein, γ-globin,β-interferon, γ-glutamyl transferase, human ubiquitin C (UBC), mousemammary tumor virus (MMTV), Rous sarcoma virus,glyceraldehyde-3-phosphate dehydrogenase, β-actin, metallothionein II(MT II), amylase, human EF1α, cathepsin, MI muscarinic receptor,retroviral LTR (e.g. human T-cell leukemia virus HTLV), AAV ITR,interleukin-2, collagenase, platelet-derived growth factor, adenovirusE2, stromelysin, murine MX, rat insulin, glucose regulated protein 78,human immunodeficiency virus, glucose regulated protein 94,α-2-macroglobulin, MHC class I, HSP70, proliferin, immunoglobulin lightchain, T-cell receptor, HLA DQα, HLA DQβ, interleukin-2 receptor, MHCclass II, prealbumin (transthyretin), elastase I, albumin, c-fos, neuralcell adhesion molecule (NCAM), H2B histone, rat growth hormone, humanserum amyloid (SAA), muscle creatinine kinase, troponin I (TN I), andGibbon Ape Leukemia Virus (GALV). In some embodiments, the promoter maybe an inducible promoter or a constitutive promoter. Additional examplesof promoters are known in the art.

In some examples, the vectors provided herein further include a poly(A)sequence, which is operably linked and positioned 3′ to the sequenceencoding the single-chain chimeric polypeptide, the single-chainchimeric antigen receptor, or any of the polypeptides that make up themulti-chain chimeric antigen receptor. Non-limiting examples of apoly(A) sequence include those derived from bovine growth hormone(Woychik et al., Proc. Natl. Acad. Sci. U.S.A. 81(13): 3944-3948, 1984,and U.S. Pat. No. 5,122,458), mouse-β-globin, mouse-α-globin (Orkin etal., EMBO J. 4(2): 453-456, 1985), human collagen, polyoma virus (Battet al., Mol. Cell Biol. 15(9):4783-4790, 1995), the Herpes simplex virusthymidine kinase gene (HSV TK), IgG heavy chain gene polyadenylationsignal (U.S. Patent Application Publication No. 2006/0040354), humangrowth hormone (hGH) (Szymanski et al., Mol. Therapy 15(7):1340-1347,2007), SV40 poly(A) site, e.g., SV40 late and early poly(A) site (Scheket al., Mol. Cell Biol. 12(12):5386-5393, 1992). In some embodiments,the poly(A) sequence includes a highly conserved upstream element(AATAAA). The this AATAAA sequence can, e.g., be substituted with otherhexanucleotide sequences with homology to AATAAA which are capable ofsignaling polyadenylation, including, e.g., ATTAAA, AGTAAA, CATAAA,TATAAA, GATAAA, ACTAAA, AATATA, AAGAAA, AATAAT, AAAAAA, AATGAA, AATCAA,AACAAA, AATCAA, AATAAC, AATAGA, AATTAA, and AATAAG. See, e.g., WO06012414 A2).

A poly(A) sequence can, e.g., be a synthetic polyadenylation site. See,e.g., Levitt el al, Genes Dev. 3(7): 1019-1025, 1989). In some examples,a poly(A) sequence can be the polyadenylation signal of solubleneuropilin-1: AAATAAAATACGAAATG. Additional examples of poly(A)sequences are known in the art. Additional examples and aspects ofvectors are also known in the art.

Methods of Introducing a Nucleic Acid or Vectors into a Mammalian Cell

A variety of different methods known in the art can be used to introduceany of the nucleic acids and vectors disclosed herein into a mammaliancell (e.g., any of the mammalian cells described herein, e.g., any ofthe T cells (e.g., human T cells) described herein). Non-limitingexamples of methods that can be used to introduce a nucleic acid orvector into a mammalian cell include lipofection, transfection,electroporation, microinjection, calcium phosphate transfection,dendrimer-based transfection, cationic polymer transfection, cellsqueezing, sonoporation, optical transfection, impalection, hydrodynamicdelivery, magnetofection, viral transduction (e.g., adenoviral andlentiviral transduction), and nanoparticle transfection. Additionalmethods of introducing a nucleic acid or vector into a mammalian cellare known in the art.

Mammalian Cells

Also provided herein are mammalian cells that include any of the nucleicacids or vectors described herein. In some embodiments, the mammaliancell is previously obtained from a subject (e.g., a human subject, e.g.,a human subject identified or diagnosed as having a cancer).

Non-limiting examples of mammalian cells include a T cell (e.g., a humanT cell). Non limiting examples of T cells (e.g., human T cells) include,e.g., an immature thymocyte, a peripheral blood lymphocyte, a naïve Tcell, a pluripotent T_(H) cell precursor, a lymphoid progenitor cell, aT_(reg) cell, a memory T cell, a T_(H)17 cell, a T_(H)22 cell, a T_(H)9cell, a T_(H)2 cell, a T_(H)1 cell, a T_(H)3 cell, γδ T cell, an αβ Tcell, a Treg cell, and a tumor-infiltrating T cell. Additional examplesof a T cell (e.g., a human T cell) include a CD8⁺ T cell, a CD4⁺ T cell,and natural killer T cell. Additional examples of mammalian cellsinclude a mast cell, a macrophage, a neutrophil, a dendritic cell, abasophil, an eosinophil, and a natural killer cell.

Hormones and Hormone Analogues

Non-limiting examples of hormones include estrogen (17β-estradiol),progesterone, testosterone, and dihydrotestosterone. Non-limitingexamples of hormone analogues include estrogen hormone analogues,progesterone hormone analogues, and androgen hormone analogues.

Non-limiting examples of estrogen hormone analogues include: afimoxifene(also known as 4-hydroxytamoxifen, 4-OHT, 4-HT, and OHTAM); selectiveestrogen receptor modulators (SERMs), such as clomifene, ormeloxifene,raloxifene, tamoxifen, toremifene, lasofoxifene, and ospemifene;estradiol; estrone; raloxifene; estriol; genistein; phytoestrogens(e.g., isoflavones, such as genistein, daidzein, formononetin, andglycitein; coumestans, such as coumestrol and repensol andtrifoliol; andlignans, such as lariciresinol, matairesinol, pinoresinol,secoisolariciresinol, podophyllotoxin, and steganacin); andxenoestrogens (e.g., diethylstilbestrol, ethinyl estradiol, butylatedhydroxyanisole, erythrosine, camphors (e.g., 4-methylbenzylidenecamphor), parabens (e.g., methylparaben, ethylparaben andpropylparaben), atrazine, dichlorodiphenyldichloroethylene,dichlorodiphenyltrichloroethane, methoxychlor, dieldrin, endosulfan,heptachlor, lindane, phenols (e.g., bisphenol A and nonylphenol),biphenyls (e.g., monochlorobiphenyl and dichlorobiphenyl), phthalates(e.g., di-2-ethylhexyl phthalate, diisodecyl phthalate and diisononylphthalate), and metalloestrogens). In some embodiments, an estrogenhormone analogue can be, e.g., mifepristone (also known as RU-486). Insome embodiments, an estrogen hormone analogue can be a selectiveprogesterone receptor modulator (SPRM), such as onapristone (ZK98299),lonaprisan (ZK230211, BAY86-5044), APR19, EC304, WAY-255348, ORG31710,asoprisnil (J867), telapristone (Proellex, CDB-4124), and CDB-2914(ulipristal acetates).

Non-limiting examples of progesterone hormone analogues includeselective progesterone receptor modulators (SPRMs), such as onapristone(ZK98299), lonaprisan (ZK230211, BAY86-5044), APR19, EC304, WAY-255348,ORG31710, asoprisnil (J867), telapristone (Proellex, CDB-4124), andCDB-2914 (ulipristal acetates).

Non-limiting examples of androgen hormone analogues include enobosarm(also known as ostarine, MK-2866, GTx-024, and S-22), BMS-564,929,LGD-4033 (ligandrol), AC-262,356, JNJ-28330835, LGD-2226, LGD-3303,S-40503, S-23, RAD140, acetothiolutamide, Andarine, LG-121071,TFM-4AS-1, and YK-11.

As can be appreciated in the art, estrogen or an estrogen hormoneanalogue can be used in together with a single-chain chimericpolypeptide, a single-chain chimeric antigen receptor, or a multi-chainchimeric antigen receptor that includes an estrogen receptor ligandbinding domain.

Compositions and Kits

Also provided herein are compositions (e.g., pharmaceuticalcompositions) that include any of the nucleic acids, vectors, sets ofnucleic acids, sets of vectors, or mammalian cells described herein. Forexample, provided herein is a composition that includes any of thenucleic acids or sets of nucleic acids described herein, or any of thevectors or sets of vectors provided herein and a pharmaceuticallyacceptable solvent or carrier.

In some embodiments, a composition can be any of the mammalian cellsdescribed herein (e.g., any of the mammalian cells described hereinpreviously obtained from a subject, e.g., a subject identified ordiagnosed as having a cancer) comprising a nucleic acid encoding any ofthe single-chain chimeric polypeptides, single-chain chimeric antigenreceptors, or multi-chain chimeric antigen receptors described herein.In a composition including any of the mammalian cells described herein,the composition can further include a cell culture medium or apharmaceutically acceptable buffer (e.g., phosphate-buffered saline). Acomposition that includes any of the mammalian cells described hereincan be formulated for intravenous or intraarterial administration.

Also provided are kits that include one or more of any of thecompositions described herein. For example, a kit can include any of thecompositions described herein and one or more doses of a hormone orhormone analogue formulated for administration to a subject (e.g.,formulated for oral, intravenous, intramuscular, or subcutaneousadministration). In some embodiments, a kit can further includeinstructions for performing any of the methods described herein.

Methods of Inducing the Membrane Localization of a Single-Chain ChimericPolypeptide, a Single-Chain Chimeric Antigen Receptor, or a Multi-ChainChimeric Polypeptide in a Mammalian Cell

Also provided herein are methods of inducing the membrane localizationof a single-chain chimeric polypeptide (e.g., any of the single-chainchimeric polypeptides described herein), a single-chain chimeric antigenreceptor (e.g., any of the single-chain chimeric antigen receptorsdescribed herein), or a multi-chain chimeric antigen receptor (e.g., anyof the multi-chain chimeric antigen receptors described herein) in amammalian cell (e.g., any of the mammalian cells described herein) thatinclude: contacting a mammalian cell expressing a single-chain chimericpolypeptide (e.g., any of the single-chain chimeric polypeptidesdescribed herein), a single-chain chimeric antigen receptor (e.g., anyof the single-chain chimeric antigen receptors described herein), or amulti-chain chimeric antigen receptor (e.g., any of the multi-chainchimeric antigen receptors described herein) with an amount of a hormoneor a hormone analogue sufficient to induce localization of thesingle-chain chimeric polypeptide, the single-chain chimericantigen-binding receptor, or the multi-chain chimeric antigen receptorto the extracellular side of the plasma membrane of the cell,respectively.

Some embodiments of these methods further include introducing a nucleicacid encoding the single-chain chimeric polypeptide, the single-chainchimeric antigen receptor, or the multi-chain chimeric antigen receptorinto a mammalian cell (e.g., any of the mammalian cells described hereinor known in the art) to generate the mammalian cell expressing thesingle-chain chimeric polypeptide, the single-chain chimeric antigenreceptor, or the multi-chain chimeric antigen receptor, respectively. Insome examples of these methods, the mammalian cell is a T cell (e.g., aCD8+ T cell, a CD4+ T cell, a memory T cell, a Treg cell, and a naturalkiller T cell). In some examples, the mammalian cell (e.g., any of themammalian cells described herein) is a mammalian cell previouslyobtained from a subject (e.g., a subject that has been identified ordiagnosed as having a cancer, e.g., any of the cancers describedherein). Some embodiments of these methods further include obtaining themammalian cell from the subject.

In some embodiments of any of these methods, the contacting step isperformed in vitro. In some embodiments of any of these methods, thecontacting step is performed in a mammal (e.g., a mammal, e.g., a human,having a cancer, e.g., any of the cancers described herein). In someembodiments, the contacting step results in the treatment of the cancerin a mammal (e.g., a human).

Non-limiting examples of cancer that can be treated using any of themethods provided herein include: acute lymphoblastic leukemia, acutemyeloid leukemia, adrenocortical carcinoma, Kaposi sarcoma, lymphoma,anal cancer, appendix cancer, teratoid/rhabdoid tumor, basal cellcarcinoma, bile duct cancer, bladder cancer, bone cancer, brain cancer,breast cancer, bronchial tumor, carcinoid tumor, cardiac tumor, cervicalcancer, chordoma, chronic lymphocytic leukemia, chronicmyeloproliferative neoplasm, colon cancer, colorectal cancer,craniopharyngioma, bile duct cancer, endometrial cancer, ependymoma,esophageal cancer, esthesioneuroblastoma, Ewing sarcoma, eye cancer,fallopian tube cancer, gallbladder cancer, gastric cancer,gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, germcell tumor, hairy cell leukemia, head and neck cancer, heart cancer,liver cancer, hypopharngeal cancer, pancreatic cancer, kidney cancer,laryngeal cancer, chronic myelogenous leukemia, lip and oral cavitycancer, lung cancer, melanoma, Merkel cell carcinoma, mesothelioma,mouth cancer, oral cancer, osteosarcoma, ovarian cancer, penile cancer,pharyngeal cancer, prostate cancer, rectal cancer, salivary glandcancer, skin cancer, small intestine cancer, soft tissue sarcoma,gastric cancer, testicular cancer, throat cancer, thyroid cancer,urethral cancer, uterine cancer, vaginal cancer, and vulvar cancer.

In some embodiments of any of these methods, the methods result in adecrease in the tumor burden (e.g., tumor mass and/or volume) in asubject. For example, any of the methods described herein can result inat least about 1% to about 99% (e.g., about 1% to about 98%, about 96%,about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about82%, about 80%, about 78%, about 76%, about 74%, about 72%, about 70%,about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about35%, about 30%, about 25%, about 20%, about 15%, about 10%, or about 5%(inclusive); about 2% to about 99%, about 98%, about 96%, about 94%,about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about80%, about 78%, about 76%, about 74%, about 72%, about 70%, about 65%,about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about30%, about 25%, about 20%, about 15%, about 10%, or about 5%(inclusive); about 3% to about 99%, about 98%, about 96%, about 94%,about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about80%, about 78%, about 76%, about 74%, about 72%, about 70%, about 65%,about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about30%, about 25%, about 20%, about 15%, about 10%, or about 5%(inclusive); about 5% to about 99%, about 98%, about 96%, about 94%,about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about80%, about 78%, about 76%, about 74%, about 72%, about 70%, about 65%,about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about30%, about 25%, about 20%, about 15%, or about 10% (inclusive); about10% to about 99%, about 98%, about 96%, about 94%, about 92%, about 90%,about 88%, about 86%, about 84%, about 82%, about 80%, about 78%, about76%, about 74%, about 72%, about 70%, about 65%, about 60%, about 55%,about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about20%, or about 15% (inclusive); about 15% to about 99%, about 98%, about96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%,about 82%, about 80%, about 78%, about 76%, about 74%, about 72%, about70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%,about 35%, about 30%, about 25%, or about 20% (inclusive); about 20% toabout 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about88%, about 86%, about 84%, about 82%, about 80%, about 78%, about 76%,about 74%, about 72%, about 70%, about 65%, about 60%, about 55%, about50%, about 45%, about 40%, about 35%, about 30%, or about 25%(inclusive); about 25% to about 99%, about 98%, about 96%, about 94%,about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about80%, about 78%, about 76%, about 74%, about 72%, about 70%, about 65%,about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, orabout 30% (inclusive); about 30% to about 99%, about 98%, about 96%,about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about82%, about 80%, about 78%, about 76%, about 74%, about 72%, about 70%,about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, orabout 35% (inclusive); about 35% to about 99%, about 98%, about 96%,about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about82%, about 80%, about 78%, about 76%, about 74%, about 72%, about 70%,about 65%, about 60%, about 55%, about 50%, about 45%, or about 40%(inclusive); about 40% to about 99%, about 98%, about 96%, about 94%,about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about80%, about 78%, about 76%, about 74%, about 72%, about 70%, about 65%,about 60%, about 55%, about 50%, or about 45% (inclusive); about 45% toabout 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about88%, about 86%, about 84%, about 82%, about 80%, about 78%, about 76%,about 74%, about 72%, about 70%, about 65%, about 60%, about 55%, orabout 50% (inclusive); about 50% to about 99%, about 98%, about 96%,about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about82%, about 80%, about 78%, about 76%, about 74%, about 72%, about 70%,about 65%, about 60%, or about 55% (inclusive); about 55% to about 99%,about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about86%, about 84%, about 82%, about 80%, about 78%, about 76%, about 74%,about 72%, about 70%, about 65%, or about 60% (inclusive); about 60% toabout 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about88%, about 86%, about 84%, about 82%, about 80%, about 78%, about 76%,about 74%, about 72%, about 70%, or about 65% (inclusive); about 65% toabout 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about88%, about 86%, about 84%, about 82%, about 80%, about 78%, about 76%,about 74%, about 72%, or about 70% (inclusive); about 70% to about 99%,about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about86%, about 84%, about 82%, about 80%, about 78%, about 76%, about 74%,or about 72% (inclusive); about 72% to about 99%, about 98%, about 96%,about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about82%, about 80%, about 78%, about 76%, or about 74% (inclusive); about74% to about 99%, about 98%, about 96%, about 94%, about 92%, about 90%,about 88%, about 86%, about 84%, about 82%, about 80%, about 78%, orabout 76% (inclusive); about 76% to about 99%, about 98%, about 96%,about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about82%, about 80%, or about 78% (inclusive); about 78% to about 99%, about98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%,about 84%, about 82%, or about 80% (inclusive); about 80% to about 99%,about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about86%, about 84%, or about 82% (inclusive); about 82% to about 99%, about98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%,or about 84% (inclusive); about 84% to about 99%, about 98%, about 96%,about 94%, about 92%, about 90%, about 88%, or about 86% (inclusive);about 86% to about 99%, about 98%, about 96%, about 94%, about 92%,about 90%, or about 88% (inclusive); about 88% to about 99%, about 98%,about 96%, about 94%, about 92%, or about 90% (inclusive); about 90% toabout 99%, about 98%, about 96%, about 94%, or about 92% (inclusive);about 92% to about 99%, about 98%, about 96%, or about 94% (inclusive);about 94% to about 99%, about 98%, or about 96% (inclusive); or about96% to about 99% or about 98% (inclusive)) reduction in the tumor burdenin a subject (e.g., as compared to the tumor burden in the subject priorto treatment).

In some embodiments, the methods result in a decrease in the rate ofprogression of a cancer in the subject. For example, any of the methodsdescribed herein can result in at least about 1% to about 99% (e.g.,about 1% to about 98%, about 96%, about 94%, about 92%, about 90%, about88%, about 86%, about 84%, about 82%, about 80%, about 78%, about 76%,about 74%, about 72%, about 70%, about 65%, about 60%, about 55%, about50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%,about 15%, about 10%, or about 5% (inclusive); about 2% to about 99%,about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about86%, about 84%, about 82%, about 80%, about 78%, about 76%, about 74%,about 72%, about 70%, about 65%, about 60%, about 55%, about 50%, about45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%,about 10%, or about 5% (inclusive); about 3% to about 99%, about 98%,about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about84%, about 82%, about 80%, about 78%, about 76%, about 74%, about 72%,about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%,or about 5% (inclusive); about 5% to about 99%, about 98%, about 96%,about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about82%, about 80%, about 78%, about 76%, about 74%, about 72%, about 70%,about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about35%, about 30%, about 25%, about 20%, about 15%, or about 10%(inclusive); about 10% to about 99%, about 98%, about 96%, about 94%,about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about80%, about 78%, about 76%, about 74%, about 72%, about 70%, about 65%,about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about30%, about 25%, about 20%, or about 15% (inclusive); about 15% to about99%, about 98%, about 96%, about 94%, about 92%, about 90%, about 88%,about 86%, about 84%, about 82%, about 80%, about 78%, about 76%, about74%, about 72%, about 70%, about 65%, about 60%, about 55%, about 50%,about 45%, about 40%, about 35%, about 30%, about 25%, or about 20%(inclusive); about 20% to about 99%, about 98%, about 96%, about 94%,about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about80%, about 78%, about 76%, about 74%, about 72%, about 70%, about 65%,about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about30%, or about 25% (inclusive); about 25% to about 99%, about 98%, about96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%,about 82%, about 80%, about 78%, about 76%, about 74%, about 72%, about70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%,about 35%, or about 30% (inclusive); about 30% to about 99%, about 98%,about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about84%, about 82%, about 80%, about 78%, about 76%, about 74%, about 72%,about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about40%, or about 35% (inclusive); about 35% to about 99%, about 98%, about96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%,about 82%, about 80%, about 78%, about 76%, about 74%, about 72%, about70%, about 65%, about 60%, about 55%, about 50%, about 45%, or about 40%(inclusive); about 40% to about 99%, about 98%, about 96%, about 94%,about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about80%, about 78%, about 76%, about 74%, about 72%, about 70%, about 65%,about 60%, about 55%, about 50%, or about 45% (inclusive); about 45% toabout 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about88%, about 86%, about 84%, about 82%, about 80%, about 78%, about 76%,about 74%, about 72%, about 70%, about 65%, about 60%, about 55%, orabout 50% (inclusive); about 50% to about 99%, about 98%, about 96%,about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about82%, about 80%, about 78%, about 76%, about 74%, about 72%, about 70%,about 65%, about 60%, or about 55% (inclusive); about 55% to about 99%,about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about86%, about 84%, about 82%, about 80%, about 78%, about 76%, about 74%,about 72%, about 70%, about 65%, or about 60% (inclusive); about 60% toabout 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about88%, about 86%, about 84%, about 82%, about 80%, about 78%, about 76%,about 74%, about 72%, about 70%, or about 65% (inclusive); about 65% toabout 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about88%, about 86%, about 84%, about 82%, about 80%, about 78%, about 76%,about 74%, about 72%, or about 70% (inclusive); about 70% to about 99%,about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about86%, about 84%, about 82%, about 80%, about 78%, about 76%, about 74%,or about 72% (inclusive); about 72% to about 99%, about 98%, about 96%,about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about82%, about 80%, about 78%, about 76%, or about 74% (inclusive); about74% to about 99%, about 98%, about 96%, about 94%, about 92%, about 90%,about 88%, about 86%, about 84%, about 82%, about 80%, about 78%, orabout 76% (inclusive); about 76% to about 99%, about 98%, about 96%,about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about82%, about 80%, or about 78% (inclusive); about 78% to about 99%, about98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%,about 84%, about 82%, or about 80% (inclusive); about 80% to about 99%,about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about86%, about 84%, or about 82% (inclusive); about 82% to about 99%, about98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%,or about 84% (inclusive); about 84% to about 99%, about 98%, about 96%,about 94%, about 92%, about 90%, about 88%, or about 86% (inclusive);about 86% to about 99%, about 98%, about 96%, about 94%, about 92%,about 90%, or about 88% (inclusive); about 88% to about 99%, about 98%,about 96%, about 94%, about 92%, or about 90% (inclusive); about 90% toabout 99%, about 98%, about 96%, about 94%, or about 92% (inclusive);about 92% to about 99%, about 98%, about 96%, or about 94% (inclusive);about 94% to about 99%, about 98%, or about 96% (inclusive); or about96% to about 99% or about 98% (inclusive)) reduction in the rate ofprogression of a cancer in a subject (e.g., as compared to the rate ofprogression of a cancer in the subject prior to treatment or in acontrol subject or a control population of subjects having the samecancer and administered no treatment or a different treatment).

In some embodiments of any of these methods, the methods result in anincrease in the time of survival of a cancer in a subject. For example,any of the methods described herein can result in an about 1% to about800% (e.g., about 1% to about 750%, about 700%, about 650%, about 600%,about 550%, about 500%, about 450%, about 400%, about 350%, about 300%,about 250%, about 200%, about 150%, about 100%, about 80%, about 60%,about 40%, about 20%, about 10%, or about 5% (inclusive); about 5% toabout 800%, about 750%, about 700%, about 650%, about 600%, about 550%,about 500%, about 450%, about 400%, about 350%, about 300%, about 250%,about 200%, about 150%, about 100%, about 80%, about 60%, about 40%,about 20%, or about 10% (inclusive); about 10% to about 800%, about750%, about 700%, about 650%, about 600%, about 550%, about 500%, about450%, about 400%, about 350%, about 300%, about 250%, about 200%, about150%, about 100%, about 80%, about 60%, about 40%, or about 20%(inclusive); about 20% to about 800%, about 750%, about 700%, about650%, about 600%, about 550%, about 500%, about 450%, about 400%, about350%, about 300%, about 250%, about 200%, about 150%, about 100%, about80%, about 60%, or about 40% (inclusive); about 40% to about 800%, about750%, about 700%, about 650%, about 600%, about 550%, about 500%, about450%, about 400%, about 350%, about 300%, about 250%, about 200%, about150%, about 100%, about 80%, or about 60% (inclusive); about 60% toabout 800%, about 750%, about 700%, about 650%, about 600%, about 550%,about 500%, about 450%, about 400%, about 350%, about 300%, about 250%,about 200%, about 150%, about 100%, about 80% (inclusive); about 80% toabout 800%, about 750%, about 700%, about 650%, about 600%, about 550%,about 500%, about 450%, about 400%, about 350%, about 300%, about 250%,about 200%, about 150%, or about 100% (inclusive); about 100% to about800%, about 750%, about 700%, about 650%, about 600%, about 550%, about500%, about 450%, about 400%, about 350%, about 300%, about 250%, about200%, or about 150% (inclusive); about 150% to about 800%, about 750%,about 700%, about 650%, about 600%, about 550%, about 500%, about 450%,about 400%, about 350%, about 300%, about 250%, or about 200%(inclusive); about 200% to about 800%, about 750%, about 700%, about650%, about 600%, about 550%, about 500%, about 450%, about 400%, about350%, about 300%, or about 250% (inclusive); about 250% to about 800%,about 750%, about 700%, about 650%, about 600%, about 550%, about 500%,about 450%, about 400%, about 350%, or about 300% (inclusive); about300% to about 800%, about 750%, about 700%, about 650%, about 600%,about 550%, about 500%, about 450%, about 400%, or about 350%(inclusive); about 350% to about 800%, about 750%, about 700%, about650%, about 600%, about 550%, about 500%, about 450%, or about 400%(inclusive); about 400% to about 800%, about 750%, about 700%, about650%, about 600%, about 550%, about 500%, or about 450% (inclusive);about 450% to about 800%, about 750%, about 700%, about 650%, about600%, about 550%, or about 500% (inclusive); about 500% to about 800%,about 750%, about 700%, about 650%, about 600%, or about 550%(inclusive); about 550% to about 800%, about 750%, about 700%, about650%, or about 600% (inclusive); about 600% to about 800%, about 750%,about 700%, or about 650% (inclusive); about 650% to about 800%, about750%, or about 700% (inclusive); about 700% to about 800% or about 750%(inclusive); or about 750% to about 800% (inclusive)) increase in thetime of survival of a cancer in a subject (e.g., as compared to the timeof survival for a control subject or a population of control subjectshaving the same cancer and receiving no treatment or a differenttreatment).

Methods of Reversibly Altering the Membrane Localization of aSingle-Chain Chimeric Polypeptide, a Single-Chain Chimeric AntigenReceptor, or a Multi-Chain Chimeric Polypeptide in a Mammalian Cell

Also provided herein are methods of reversibly altering the membranelocalization of a single-chain chimeric polypeptide (e.g., any of thesingle-chain polypeptides described herein), a single-chain chimericantigen receptor (e.g., any of the single-chain chimeric antigenreceptors described herein), or a multi-chain chimeric antigen receptor(e.g., any of the multi-chain chimeric antigen receptors describedherein) in a mammalian cell that include: (a) contacting a mammaliancell (e.g., any of the mammalian cells described herein) expressing asingle-chain chimeric polypeptide (e.g., any of the single-chainpolypeptides described herein), a single-chain chimeric antigen receptor(e.g., any of the single-chain chimeric antigen receptors describedherein), or a multi-chain chimeric antigen receptor (e.g., any of themulti-chain chimeric antigen receptors described herein) with an amountof a hormone or a hormone analogue sufficient to induce localization ofthe single-chain chimeric polypeptide, the single-chain chimeric antigenreceptor, or the multi-chain chimeric antigen receptor to theextracellular side of the plasma membrane of the cell, respectively; and(b) contacting the mammalian cell with a reduced amount of the hormoneor the hormone analogue that results in a decreased level of thesingle-chain chimeric polypeptide, the single-chain chimeric antigenreceptor, or the multi-chain chimeric antigen receptor on theextracellular side of the plasma membrane of the mammalian cell (e.g.,as compared to the level on the extracellular side of the plasmamembrane in step (a)). In some embodiments, the decreased level in step(b) is at least a 5% decrease, at least a 10% decrease, at least a 15%decrease, at least a 20% decrease, at least a 25% decrease, at least a30% decrease, at least a 35% decrease, at least a 40% decrease, at leasta 45% decrease, at least a 50% decrease, at least a 55% decrease, atleast a 60% decrease, at least a 65% decrease, at least a 70% decrease,at least a 75% decrease, at least a 80% decrease, at least a 85%decrease, at least a 90% decrease, at least a 95% decrease, or at leasta 99% decrease in the level of the single-chain chimeric polypeptide,the single-chain chimeric antigen receptor, or the multi-chain chimericantigen receptor on the extracellular side of the plasma membrane instep (a). In some embodiments, the decreased level in step (b) is a 100%decrease in the level of the single-chain chimeric polypeptide, thesingle-chain chimeric antigen receptor, or the multi-chain chimericantigen receptor on the extracellular side of the plasma membrane instep (a). In some embodiments, the mammalian cell is not contacted withany hormone or hormone analogue hormone in step (b). Some embodiments ofthese methods further include: (c) contacting the mammalian cell (e.g.,any of the mammalian cells described herein) with an increased amount ofthe hormone or the hormone analogue that results in an increased levelof the single-chain chimeric polypeptide, the single-chain chimericantigen receptor, or the multi-chain chimeric antigen receptor on theextracellular side of the plasma membrane of the cell as compared to thelevel of in step (b).

Some embodiments of any of these methods further include introducing anucleic acid encoding the single-chain chimeric polypeptide into amammalian cell (e.g., any of the mammalian cells described herein) togenerate the mammalian cell expressing the single-chain chimericpolypeptide, the single-chain chimeric antigen receptor, or themulti-chain chimeric antigen receptor. In some embodiments of any of themethods described herein, the mammalian cell is a T cell (e.g., a humanT cell). In some embodiments, the T cell (e.g., human T cell) isselected from the group of a CD8⁺ T cell, a CD4⁺ T cell, a memory Tcell, a Treg cell, and natural killer T cell. In some embodiments, themammalian cell is a mammalian cell previously obtained from a subject(e.g., a human). In some examples, the subject (e.g., human) has beenidentified or diagnosed as having a cancer (e.g., any of the types ofcancer described herein). Some embodiments further include obtaining themammalian cell from the subject.

In some embodiments of any of the methods described herein, thecontacting step is performed in vitro. In some embodiments, thecontacting step is performed in a mammal (e.g., in a subject). In someembodiments, the contacting step results in the treatment of the cancerin a mammal (e.g., a human).

Non-limiting examples of cancer that can be treated using any of themethods provided herein include: acute lymphoblastic leukemia, acutemyeloid leukemia, adrenocortical carcinoma, Kaposi sarcoma, lymphoma,anal cancer, appendix cancer, teratoid/rhabdoid tumor, basal cellcarcinoma, bile duct cancer, bladder cancer, bone cancer, brain cancer,breast cancer, bronchial tumor, carcinoid tumor, cardiac tumor, cervicalcancer, chordoma, chronic lymphocytic leukemia, chronicmyeloproliferative neoplasm, colon cancer, colorectal cancer,craniopharyngioma, bile duct cancer, endometrial cancer, ependymoma,esophageal cancer, esthesioneuroblastoma, Ewing sarcoma, eye cancer,fallopian tube cancer, gallbladder cancer, gastric cancer,gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, germcell tumor, hairy cell leukemia, head and neck cancer, heart cancer,liver cancer, hypopharngeal cancer, pancreatic cancer, kidney cancer,laryngeal cancer, chronic myelogenous leukemia, lip and oral cavitycancer, lung cancer, melanoma, Merkel cell carcinoma, mesothelioma,mouth cancer, oral cancer, osteosarcoma, ovarian cancer, penile cancer,pharyngeal cancer, prostate cancer, rectal cancer, salivary glandcancer, skin cancer, small intestine cancer, soft tissue sarcoma,gastric cancer, testicular cancer, throat cancer, thyroid cancer,urethral cancer, uterine cancer, vaginal cancer, and vulvar cancer.

Methods of Controlling the Toxicity of a Single-Chain ChimericPolypeptide, a Single-Chain Chimeric Antigen Receptor, or a Multi-ChainChimeric Polypeptide in a Mammalian Cell by Reversibly Altering MembraneLocalization of the Same

In some embodiments, compositions and/or methods disclosed herein can beused to selectively kill cancer cells in a subject (e.g., a human) inthe presence of a hormone or a hormone analogue which binds to an LBDpresent in the polypeptide (e.g., a single-chain chimeric polypeptide, asingle-chain chimeric antigen receptor, or a multi-chain chimericantigen receptor). In some embodiments, in the presence of the hormoneor hormone analogue, the polypeptide (e.g., the single-chain chimericpolypeptide, the single-chain chimeric antigen receptor, or themulti-chain chimeric antigen receptor) localizes to the extracellularsurface of a mammalian cell, presenting its antigen binding domain(e.g., an scFv or any other antigen binding domain described herein orknown in the art) on the exterior of the cell. The polypeptide can thenrecognize the antigen to which it binds, and thus can selectively kill atarget cell expressing the antigen on its surface.

In some embodiments, localization of the polypeptide (e.g., thesingle-chain chimeric polypeptide, the single-chain chimeric antigenreceptor, or the multi-chain chimeric antigen receptor) on theextracellular surface can cause toxicity in the subject. Such toxicitycan be caused by, for example, a cytokine storm (also referred to ashypercytokinemia or cytokine release syndrome). In some embodiments, asubject experiencing toxicity due hormone- or hormone analogue-inducedlocalization of the polypeptide to the extracellular surface can betreated by reducing the dose of the hormone or hormone analogueadministered to the subject, or eliminating hormone or hormone analoguetreatment altogether. Such reduction in dose or elimination of thehormone or hormone analgue treatment can cause the polypeptide to beremoved from the cell surface and reduce or eliminate presentation ofthe antigen binding domain on the cell surface, thus reducing oreliminating toxicity in the subject.

Exemplary symptoms of toxicity include, without limitation, high fever,swelling, chills, hypotension, tachycardia, asthenia, headache, rash,scratchy throat, dyspnea, redness, extreme fatigue, nausea, and cerebraledema (swelling in the brain). In some embodiments, toxicity in asubject can be determined by assessing the presence or severity of oneor more of these symptoms in a subject following administration of oneor more doses of the hormone or hormone analogue to the subject. In someembodiments, one or more of these or other symptoms associated withtoxicity can be determined after introducing into a subject a mammaliancell (e.g., a T cell) that includes a single-chain chimeric polypeptide(e.g., any of the single-chain polypeptides described herein), asingle-chain chimeric antigen receptor (e.g., any of the single-chainchimeric antigen receptors described herein), or a multi-chain chimericantigen receptor (e.g., any of the multi-chain chimeric antigenreceptors described herein) having a LBD, and a hormone or hormoneanalogue that binds the LBD. In some embodiments, the determined levelof one or more of these or other symptoms associated with toxicity canbe compared to a baseline level that is associated with lack of atoxicity or a reduced level of toxicity. Such a baseline level can be,for example, the level of one or more of these or other symptoms in thesubject prior to administration of the mammalian cell, the hormone orhormone analogue, or both. Additionally or alternatively, such abaseline level can be the level of one or more of these or othersymptoms in a different subject that has not been administered themammalian cell, the hormone or hormone analogue, or both. Those ofordinary skill in the art will be aware of suitable clinical andlaboratory methods to determine the level of one or more symptomsassociated with toxicity.

In some embodiments, toxicity can be the result of a cytokine storm.Both pro-inflammatory cytokines and anti-inflammatory cytokines areelevated in the serum of subjects experiencing a cytokine storm.Non-limiting examples of cytokines that are elevated in the serum ofsubjects experiencing a cytokine storm include tumor necrosisfactor-alpha, IFNγ, IL-10, IL-1β, IL-2, IL-6, IL-8, and IL-10,granulocyte macrophage—colony-stimulating factor (GM-CSF), and IL-5. Insome embodiments, one or more of these or other cytokines associatedwith a cytokine storm are determined after introducing into a subject amammalian cell (e.g., a T cell) that includes a single-chain chimericpolypeptide (e.g., any of the single-chain polypeptides describedherein), a single-chain chimeric antigen receptor (e.g., any of thesingle-chain chimeric antigen receptors described herein), or amulti-chain chimeric antigen receptor (e.g., any of the multi-chainchimeric antigen receptors described herein) having a LBD, and a hormoneor hormone analogue that binds the LBD. In some embodiments, thedetermined level of one or more of these or other cytokines associatedwith a cytokine storm can be compared to a baseline level that isassociated with lack of a cytokine storm or a reduced level of acytokine storm. Such a baseline level can be, for example, the level ofone or more of these or other cytokines in the subject prior toadministration of the mammalian cell, the hormone or hormone analogue,or both. Additionally or alternatively, such a baseline level can be thelevel of one or more of these or other cytokines in a different subjectthat has not been administered the mammalian cell, the hormone orhormone analogue, or both. The level of a cytokine (e.g., the level ofcytokine after administering to a subject the mammalian cell and thehormone or hormone analogue and/or the baseline level of a cytokine),can be determined by any of a variety of methods and techniques,including, without limitation, ELISAs, Western blots, antibody arraypanels, multiplex arrays, Elispot assays, immunohistochemistry, or anyof the variety of commercial assays available to determine cytokinelevels.

In some embodiments, methods of treating cancer (e.g., any of thevariety of cancers described herein) in a subject (e.g., a mammal, e.g.,a human) are provided. In some embodiments, methods of treating cancerinclude: (a) administering to a subject a mammalian cell (e.g., a Tcell) that includes a nucleic acid encoding a single-chain chimericpolypeptide (e.g., any of the single-chain polypeptides describedherein), a single-chain chimeric antigen receptor (e.g., any of thesingle-chain chimeric antigen receptors described herein), or amulti-chain chimeric antigen receptor (e.g., any of the multi-chainchimeric antigen receptors described herein) having a hormone receptorLBD, (b) administering to the subject a hormone or hormone analogue thatbinds the LBD, (c) determining the level of one or more symptomsassociated with toxicity and/or the level of one or more cytokinesassociated with a cytokine storm, (d) comparing the determined level ofone or more symptoms associated with toxicity and/or the level of one ormore cytokines associated with a cytokine storm to a baseline level ofthe symptom or cytokine, and (e) reducing the amount of the hormone orhormone analogue administered to the subject, such that the determinedlevel of one or more symptoms associated with toxicity and/or the levelof one or more cytokines associated with a cytokine storm is reduced. Insome embodiments the level of the hormone or hormone analogueadministered to the subject is reduced in step (e) by at least 5%, atleast 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, or at least 99%. In someembodiments, the level of the hormone or hormone analogue administeredto the subject is reduced in step (e) by 100% (e.g., no mammalian cellis administered to the subject in step (e)). In some embodiments, afteradministering the hormone or hormone analogue in step (b), thesingle-chain chimeric polypeptide, the single-chain chimeric antigenreceptor, and/or the multi-chain chimeric antigen receptor localizes theextracellular surface of the mammalian cell in a reversible manner. Insome embodiments, after reducing the amount of the hormone or hormoneanalogue administered to the subject in step (e), the single-chainchimeric polypeptide, the single-chain chimeric antigen receptor, and/orthe multi-chain chimeric antigen receptor is removed from theextracellular surface, thus reducing the toxicity (e.g., one or moresymptoms associated with toxicity and/or one or more cytokinesassociated with a cytokine storm). In some embodiments, after reducingthe amount of the hormone or hormone analogue administered to thesubject in step (e), the toxicity (e.g., one or more symptoms associatedwith toxicity and/or one or more cytokines associated with a cytokinestorm) is reduced by at least 5%, at least 10%, at least 15%, at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, or at least 99%. In some embodiments, after reducing the amount ofthe hormone or hormone analogue administered to the subject in step (e),the toxicity (e.g., one or more symptoms associated with toxicity and/orone or more cytokines associated with a cytokine storm) is reduced by100% (e.g., the subject experiences no toxicity, or only a baselinelevel of toxicity). In some embodiments, after reducing the amount ofthe hormone or hormone analogue administered to the subject in step (e),the toxicity (e.g., one or more symptoms associated with toxicity and/orone or more cytokines associated with a cytokine storm) normalizestowards a baseline level of toxicity (e.g., a level that is associatedwith lack of a cytokine storm or a reduced level of a cytokine storm).For example, after reducing the amount of the hormone or hormoneanalogue administered to the subject in step (e), the toxicity cannormalize to a level that is less than 10-fold higher, 9-fold higher,8-fold higher, 7-fold higher, 6-fold higher, 5-fold higher, 4-foldhigher, 3-fold higher, 2-fold higher, or less than the baseline level oftoxicity.

In some embodiments, a subject can be treated by administering to thesubject a mammalian cell (e.g., a T cell) that includes a single-chainchimeric polypeptide (e.g., any of the single-chain polypeptidesdescribed herein), a single-chain chimeric antigen receptor (e.g., anyof the single-chain chimeric antigen receptors described herein), and/ora multi-chain chimeric antigen receptor (e.g., any of the multi-chainchimeric antigen receptors described herein) having a LBD and a hormoneor hormone analogue that binds the LBD. The level of toxicity in thesubject can be monitored, and toxicity can be reduced by any of themethods described herein, e.g., by reducing the level of the hormone orhormone analogue. After toxicity has been reduced (e.g., to a baselinelevel, or a level above the baseline level that is sufficiently low),the level of the hormone or hormone analogue can be subsequentlyincreased to increase the efficacy of treatment. In some embodiments,the level of the hormone or hormone analogue is decreased andsubsequently increased multiple times to control toxicity while stillproviding an effective course of treatment. In some embodiments, thelevel of the hormone is subsequently increased to 100% of the level atwhich the hormone or hormone analogue was originally administered. Insome embodiments, the level of the hormone or hormone analogue issubsequently increased to a level that is at least 5%, at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or at least 99% of the level at which thehormone or hormone analogue was originally administered. In someembodiments, the level of the hormone or hormone analogue issubsequently increased to a level that is higher than the level at whichthe hormone or hormone analogue was originally administered. Forexample, the subsequently administered hormone or hormone analogue canbe administered at a level that is at least 100%, at least 110%, atleast 120%, at least 130%, at least 140%, at least 150%, at least 160%,at least 170%, at least 180%, at least 190%, at least 200%, at least300%, at least 400%, or at least 500% of the level at which the hormoneor hormone analogue was originally administered.

In some embodiments, toxicity arising after administering a firsthormone or hormone analogue to a subject can be reduced by administeringa second hormone or hormone analogue to the subject. For example, thesecond hormone or hormone analogue can have a reduced affinity for theLBD present in the single-chain chimeric polypeptide, the single-chainchimeric antigen receptor, and/or the multi-chain chimeric antigenreceptor, such that plasma membrane localization of the single-chainchimeric polypeptide, the single-chain chimeric antigen receptor, and/orthe multi-chain chimeric antigen receptor is reduced as compared to theplasma membrane localization facilitated by the first hormone or hormoneanalogue. In some embodiments, the second hormone or hormone analoguecan be less effective than the first hormone or hormone analogue atfacilitating plasma membrane localization of the single-chain chimericpolypeptide, the single-chain chimeric antigen receptor, and/or themulti-chain chimeric antigen receptor. In some embodiments, the secondhormone or hormone analogue competes with the first hormone or hormoneanalogue for binding to the LBD.

In some embodiments, in addition to or as an alternative to reducing thelevel of hormone or hormone analogue to reduce toxicity, toxicity can befurther reduced by administering a composition that inactivates orsequesters the hormone or hormone analogue. For example, exogenous LBDcan be administered to the subject (e.g., as a pharmaceuticallyacceptable formulation) to sequester the hormone or hormone analoguethat was previously administered to the subject. As another example, acomposition that inactivates, degrades, or otherwise sequesters theadministered hormone or hormone analogue can be administered to thesubject. A non-limiting example of such a composition is an antibody orantibody fragment that binds the administered hormone or hormoneanalogue and renders it unable to penetrate a cell membrane, renders itunable to bind the LBD, or facilitates its degradation.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Example 1—Generation of Constructs

A canonical chimeric antigen receptor (“Canonical CAR”, FIG. 1, left)was modified to include the estrogen receptor alpha (“ERa”) ligandbinding domain (“ERa-LBD,” in green and indicated by arrows) at variouslocations within the protein. The ERa-LBD was introduced at differentpositions in the CAR (FIG. 1, right) to create different versions of theERa-LBD, denoted as: “v.2” (also denoted as “v2” or “V2”), “v.3” (alsodenoted as “v3” or “V3”), and “v.4” (also denoted as “v4” or “V4”). AnmCherry fusion was also included to allow tracking of cells containingthe engineered protein. The nucleic acid and polypeptide sequences ofv.2 (SEQ ID NOs: 30 and 31, respectively), v.3 (SEQ ID NOs: 32 and 33,respectively), and v.4 (SEQ ID NOs: 34 and 35, respectively) areprovided below.

SEQ ID NO: 30 ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCCCTGCTGCTGCATGCCGCTAGACCTGAGCAGAAGCTGATCTCCGAAGAGGACCTGGACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGATAGAGTGACCATCAGCTGCAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAACCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCAGACTGCACAGCGGCGTGCCCAGCAGATTTTCTGGCAGCGGCTCCGGCACCGACTACAGCCTGACCATCTCCAACCTGGAACAGGAAGATATCGCTACCTACTTCTGTCAGCAAGGCAACACCCTGCCCTACACCTTCGGCGGAGGCACCAAGCTGGAAATCACAGGCGGCGGAGGATCTGGCGGAGGCGGAAGTGGCGGAGGGGGATCTGAAGTGAAACTGCAGGAAAGCGGCCCTGGCCTGGTGGCCCCATCTCAGTCTCTGAGCGTGACCTGTACCGTGTCCGGCGTGTCCCTGCCTGACTATGGCGTGTCCTGGATCAGACAGCCCCCCAGAAAGGGCCTGGAATGGCTGGGAGTGATCTGGGGCAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTGACCATCATCAAGGACAACTCCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTACGGCGGCAGCTACGCCATGGACTACTGGGGCCAGGGCACAAGCGTGACCGTGTCTAGCACAACCACCCCTGCCCCTAGACCTCCAACCCCAGCCCCTACAATCGCCAGCCAGCCTCTGTCTCTGAGGCCCGAGGCTTGTAGACCAGCTGCTGGCGGAGCCGTGCACACCAGAGGACTGGATTTCGCCTGCGACATCTACATCTGGGCCCCTCTGGCCGGCACATGTGGCGTGCTGCTGCTGAGCCTCGTGATCACCCTGTACTGCGGATCCAAGCGGGGCAGAAAGAAACTGCTGTACATCTTTAAGCAGCCCTTCATGCGGCCCGTGCAGACCACCCAGGAAGAGGACGGCTGCTCCTGCAGATTCCCCGAGGAAGAAGAAGGCGGCTGCGAGCTGGGCAGCGGATCTGGCAGTGGAAGCGATAGAAGAGGCGGCAGAATGCTGAAACACAAGCGGCAGAGGGACGACGGGGAAGGCAGAGGCGAAGTGGGATCTGCCGGCGATATGAGAGCCGCCAACCTGTGGCCTAGCCCCCTGATGATCAAGCGGAGCAAGAAGAACTCCCTGGCCCTGAGCCTGACCGCCGACCAGATGGTGTCTGCCCTGCTGGATGCCGAGCCCCCCATCCTGTACAGCGAGTACGACCCCACCAGACCCTTCAGCGAGGCCAGCATGATGGGCCTGCTGACCAACCTGGCCGACCGGGAACTGGTGCACATGATCAACTGGGCCAAGCGGGTGCCCGGCTTCGTGGATCTGACACTGCACGACCAGGTGCACCTGCTGGAATGCGCTTGGCTGGAAATCCTGATGATCGGCCTCGTGTGGCGGAGCATGGAACACCCTGGCAAGCTGCTGTTCGCCCCCAACCTGCTGCTGGACCGGAACCAGGGCAAATGCGTGGAAGGCATGGTGGAAATCTTCGACATGCTGCTGGCCACCTCCAGCCGGTTCCGGATGATGAACCTGCAGGGCGAAGAGTTCGTGTGTCTGAAGTCCATCATCCTGCTGAATAGCGGCGTGTACACCTTCCTGAGCAGCACCCTGAAAAGCCTGGAAGAAAAGGACCACATCCACCGGGTGCTGGACAAGATCACCGACACCCTGATTCACCTGATGGCCAAGGCCGGACTGACCCTGCAGCAGCAGCATCAGAGACTGGCTCAGCTGCTGCTGATCCTGTCCCACATCCGGCACATGAGCAACAAGCGGATGGAACATCTGTACAGCATGAAGTGCAAGAACGTGGTGCCTCTGTTCGATCTGCTGCTGGAAATGCTGGACGCCCACAGGCTGCACGCCCCAACATCCAGATCCGGATCTGGAAGTGGCTCCCTGAGAGTGAAGTTTAGCAGAAGCGCCGACGCCCCTGCCTATCAGCAGGGACAGAACCAGCTGTATAACGAGCTGAACCTGGGCAGGCGGGAAGAGTACGACGTGCTGGATAAGAGGCGGGGCAGGGACCCTGAAATGGGCGGCAAACCCAGACGGAAGAACCCCCAGGAAGGCCTGTACAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGGCGGAGAGGCAAGGGACATGATGGCCTGTACCAGGGCCTGTCCACCGCCACCAAGGACACCTATGACGCCCTGCACATGCAGGCCCTGCCTCCAAGAGGAAGTGGATCTGGGAGCGGCTCTATGGTGTCTAAGGGGGAAGAGGACAACATGGCCATCATCAAAGAATTCATGCGGTTCAAGGTGCACATGGAAGGCTCCGTGAATGGCCACGAATTCGAGATCGAGGGGGAGGGCGAGGGCAGACCTTATGAGGGAACCCAGACCGCCAAGCTGAAAGTGACCAAGGGCGGACCCCTGCCTTTCGCCTGGGATATCCTGTCTCCCCAGTTTATGTACGGCAGCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACCTGAAGCTGAGCTTCCCTGAGGGCTTCAAGTGGGAGAGAGTGATGAATTTCGAGGACGGCGGAGTCGTGACAGTGACCCAGGATAGCTCTCTGCAGGACGGCGAGTTCATCTACAAAGTGAAGCTGCGGGGCACCAACTTCCCCAGCGACGGACCCGTGATGCAGAAAAAGACCATGGGCTGGGAGGCCAGCTCCGAGAGAATGTACCCAGAGGACGGGGCCCTGAAGGGGGAGATCAAGCAGCGGCTGAAACTGAAGGATGGCGGCCACTACGACGCAGAAGTGAAAACCACCTACAAGGCCAAGAAACCTGTGCAGCTGCCTGGCGCCTACAATGTGAACATCAAGCTGGACATTACCAGCCACAACGAGGACTACACCATCGTGGAACAGTACGAGCGGGCCGAGGGCAGGCATTCTACAGGCGGAATGGATGAACTGTATAAGTGCGTGACCGACTAG SEQ ID NO: 31MALPVTALLLPLALLLHAARPEQKLISEEDLDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCGSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGSGSGSGSDRRGGRMLKHKRQRDDGEGRGEVGSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKRMEHLYSMKCKNVVPLFDLLLEMLDAHRLHAPTSRSGSGSGSLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGSGSGSMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGG MDELYKCVTD SEQ ID NO:32 ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCCCTGCTGCTGCATGCCGCTAGACCTGAGCAGAAGCTGATCTCCGAAGAGGACCTGGACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGATAGAGTGACCATCAGCTGCAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAACCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCAGACTGCACAGCGGCGTGCCCAGCAGATTTTCTGGCAGCGGCTCCGGCACCGACTACAGCCTGACCATCTCCAACCTGGAACAGGAAGATATCGCTACCTACTTCTGTCAGCAAGGCAACACCCTGCCCTACACCTTCGGCGGAGGCACCAAGCTGGAAATCACAGGCGGCGGAGGATCTGGCGGAGGCGGAAGTGGCGGAGGGGGATCTGAAGTGAAACTGCAGGAAAGCGGCCCTGGCCTGGTGGCCCCATCTCAGTCTCTGAGCGTGACCTGTACCGTGTCCGGCGTGTCCCTGCCTGACTATGGCGTGTCCTGGATCAGACAGCCCCCCAGAAAGGGCCTGGAATGGCTGGGAGTGATCTGGGGCAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTGACCATCATCAAGGACAACTCCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTACGGCGGCAGCTACGCCATGGACTACTGGGGCCAGGGCACAAGCGTGACCGTGTCTAGCACAACCACCCCTGCCCCTAGACCTCCAACCCCAGCCCCTACAATCGCCAGCCAGCCTCTGTCTCTGAGGCCCGAGGCTTGTAGACCAGCTGCTGGCGGAGCCGTGCACACCAGAGGACTGGATTTCGCCTGCGACATCTACATCTGGGCCCCTCTGGCCGGCACATGTGGCGTGCTGCTGCTGAGCCTCGTGATCACCCTGTACTGCGGATCCAAGCGGGGCAGAAAGAAACTGCTGTACATCTTTAAGCAGCCCTTCATGCGGCCCGTGCAGACCACCCAGGAAGAGGACGGCTGCTCCTGCAGATTCCCCGAGGAAGAAGAAGGCGGCTGCGAGCTGAGATCCGGATCTGGAAGTGGCTCCCTGAGAGTGAAGTTTAGCAGAAGCGCCGACGCCCCTGCCTATCAGCAGGGACAGAACCAGCTGTATAACGAGCTGAACCTGGGCAGGCGGGAAGAGTACGACGTGCTGGATAAGAGGCGGGGCAGGGACCCTGAAATGGGCGGCAAACCCAGACGGAAGAACCCCCAGGAAGGCCTGTACAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGGCGGAGAGGCAAGGGACATGATGGCCTGTACCAGGGCCTGTCCACCGCCACCAAGGACACCTATGACGCCCTGCACATGCAGGCCCTGCCTCCAAGAGGCAGCGGATCTGGCAGTGGAAGCGATAGAAGAGGCGGCAGAATGCTGAAACACAAGCGGCAGAGGGACGACGGGGAAGGCAGAGGCGAAGTGGGATCTGCCGGCGATATGAGAGCCGCCAACCTGTGGCCTAGCCCCCTGATGATCAAGCGGAGCAAGAAGAACTCCCTGGCCCTGAGCCTGACCGCCGACCAGATGGTGTCTGCCCTGCTGGATGCCGAGCCCCCCATCCTGTACAGCGAGTACGACCCCACCAGACCCTTCAGCGAGGCCAGCATGATGGGCCTGCTGACCAACCTGGCCGACCGGGAACTGGTGCACATGATCAACTGGGCCAAGCGGGTGCCCGGCTTCGTGGATCTGACACTGCACGACCAGGTGCACCTGCTGGAATGCGCTTGGCTGGAAATCCTGATGATCGGCCTCGTGTGGCGGAGCATGGAACACCCTGGCAAGCTGCTGTTCGCCCCCAACCTGCTGCTGGACCGGAACCAGGGCAAATGCGTGGAAGGCATGGTGGAAATCTTCGACATGCTGCTGGCCACCTCCAGCCGGTTCCGGATGATGAACCTGCAGGGCGAAGAGTTCGTGTGTCTGAAGTCCATCATCCTGCTGAATAGCGGCGTGTACACCTTCCTGAGCAGCACCCTGAAAAGCCTGGAAGAAAAGGACCACATCCACCGGGTGCTGGACAAGATCACCGACACCCTGATTCACCTGATGGCCAAGGCCGGACTGACCCTGCAGCAGCAGCATCAGAGACTGGCTCAGCTGCTGCTGATCCTGTCCCACATCCGGCACATGAGCAACAAGCGGATGGAACATCTGTACAGCATGAAGTGCAAGAACGTGGTGCCTCTGTTCGATCTGCTGCTGGAAATGCTGGACGCCCACAGGCTGCACGCCCCAACATCCGGATCTGGCTCTGGAAGCGGCAGCATGGTGTCTAAGGGGGAAGAGGACAACATGGCCATCATCAAAGAATTCATGCGGTTCAAGGTGCACATGGAAGGCTCCGTGAATGGCCACGAATTCGAGATCGAGGGGGAGGGCGAGGGCAGACCTTATGAGGGAACCCAGACCGCCAAGCTGAAAGTGACCAAGGGCGGACCCCTGCCTTTCGCCTGGGATATCCTGTCTCCCCAGTTTATGTACGGCAGCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACCTGAAGCTGAGCTTCCCTGAGGGCTTCAAGTGGGAGAGAGTGATGAATTTCGAGGACGGCGGAGTCGTGACAGTGACCCAGGATAGCTCTCTGCAGGACGGCGAGTTCATCTACAAAGTGAAGCTGCGGGGCACCAACTTCCCCAGCGACGGACCCGTGATGCAGAAAAAGACCATGGGCTGGGAGGCCAGCTCCGAGAGAATGTACCCAGAGGACGGGGCCCTGAAGGGGGAGATCAAGCAGCGGCTGAAACTGAAGGATGGCGGCCACTACGACGCAGAAGTGAAAACCACCTACAAGGCCAAGAAACCTGTGCAGCTGCCTGGCGCCTACAATGTGAACATCAAGCTGGACATTACCAGCCACAACGAGGACTACACCATCGTGGAACAGTACGAGCGGGCCGAGGGCAGGCATTCTACAGGCGGAATGGATGAACTGTATAAGTGCGTGACCGACTAG SEQ ID NO: 33MALPVTALLLPLALLLHAARPEQKLISEEDLDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCGSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRSGSGSGSLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGSGSGSDRRGGRMLKHKRQRDDGEGRGEVGSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKRMEHLYSMKCKNVVPLFDLLLEMLDAHRLHAPTSGSGSGSGSMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGG MDELYKCVTD SEQ ID NO:34 ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCCCTGCTGCTGCATGCCGCTAGACCTGAGCAGAAGCTGATCTCCGAAGAGGACCTGGACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGATAGAGTGACCATCAGCTGCAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAACCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCAGACTGCACAGCGGCGTGCCCAGCAGATTTTCTGGCAGCGGCTCCGGCACCGACTACAGCCTGACCATCTCCAACCTGGAACAGGAAGATATCGCTACCTACTTCTGTCAGCAAGGCAACACCCTGCCCTACACCTTCGGCGGAGGCACCAAGCTGGAAATCACAGGCGGCGGAGGATCTGGCGGAGGCGGAAGTGGCGGAGGGGGATCTGAAGTGAAACTGCAGGAAAGCGGCCCTGGCCTGGTGGCCCCATCTCAGTCTCTGAGCGTGACCTGTACCGTGTCCGGCGTGTCCCTGCCTGACTATGGCGTGTCCTGGATCAGACAGCCCCCCAGAAAGGGCCTGGAATGGCTGGGAGTGATCTGGGGCAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTGACCATCATCAAGGACAACTCCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTACGGCGGCAGCTACGCCATGGACTACTGGGGCCAGGGCACAAGCGTGACCGTGTCTAGCACAACCACCCCTGCCCCTAGACCTCCAACCCCAGCCCCTACAATCGCCAGCCAGCCTCTGTCTCTGAGGCCCGAGGCTTGTAGACCAGCTGCTGGCGGAGCCGTGCACACCAGAGGACTGGATTTCGCCTGCGACATCTACATCTGGGCCCCTCTGGCCGGCACATGTGGCGTGCTGCTGCTGAGCCTCGTGATCACCCTGTACTGCGGATCCGGCAGCGGATCTGGCAGTGGAAGCGATAGAAGAGGCGGCAGAATGCTGAAACACAAGCGGCAGAGGGACGACGGGGAAGGCAGAGGCGAAGTGGGATCTGCCGGCGATATGAGAGCCGCCAACCTGTGGCCTAGCCCCCTGATGATCAAGCGGAGCAAGAAGAACTCCCTGGCCCTGAGCCTGACCGCCGACCAGATGGTGTCTGCCCTGCTGGATGCCGAGCCCCCCATCCTGTACAGCGAGTACGACCCCACCAGACCCTTCAGCGAGGCCAGCATGATGGGCCTGCTGACCAACCTGGCCGACCGGGAACTGGTGCACATGATCAACTGGGCCAAGCGGGTGCCCGGCTTCGTGGATCTGACACTGCACGACCAGGTGCACCTGCTGGAATGCGCTTGGCTGGAAATCCTGATGATCGGCCTCGTGTGGCGGAGCATGGAACACCCTGGCAAGCTGCTGTTCGCCCCCAACCTGCTGCTGGACCGGAACCAGGGCAAATGCGTGGAAGGCATGGTGGAAATCTTCGACATGCTGCTGGCCACCTCCAGCCGGTTCCGGATGATGAACCTGCAGGGCGAAGAGTTCGTGTGTCTGAAGTCCATCATCCTGCTGAATAGCGGCGTGTACACCTTCCTGAGCAGCACCCTGAAAAGCCTGGAAGAAAAGGACCACATCCACCGGGTGCTGGACAAGATCACCGACACCCTGATTCACCTGATGGCCAAGGCCGGACTGACCCTGCAGCAGCAGCATCAGAGACTGGCTCAGCTGCTGCTGATCCTGTCCCACATCCGGCACATGAGCAACAAGCGGATGGAACATCTGTACAGCATGAAGTGCAAGAACGTGGTGCCTCTGTTCGATCTGCTGCTGGAAATGCTGGACGCCCACAGGCTGCACGCCCCAACATCCGGATCTGGCTCTGGAAGCGGCAGCAAGCGGGGCAGAAAGAAACTGCTGTACATCTTTAAGCAGCCCTTCATGCGGCCCGTGCAGACCACCCAGGAAGAGGACGGCTGCTCCTGCAGATTCCCCGAGGAAGAAGAAGGCGGCTGCGAGCTGAGATCCGGATCTGGAAGTGGCTCCCTGAGAGTGAAGTTTAGCAGAAGCGCCGACGCCCCTGCCTATCAGCAGGGACAGAACCAGCTGTATAACGAGCTGAACCTGGGCAGGCGGGAAGAGTACGACGTGCTGGATAAGAGGCGGGGCAGGGACCCTGAAATGGGCGGCAAACCCAGACGGAAGAACCCCCAGGAAGGCCTGTACAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGGCGGAGAGGCAAGGGACATGATGGCCTGTACCAGGGCCTGTCCACCGCCACCAAGGACACCTATGACGCCCTGCACATGCAGGCCCTGCCTCCAAGAGGAAGTGGATCTGGGAGCGGCTCTATGGTGTCTAAGGGGGAAGAGGACAACATGGCCATCATCAAAGAATTCATGCGGTTCAAGGTGCACATGGAAGGCTCCGTGAATGGCCACGAATTCGAGATCGAGGGGGAGGGCGAGGGCAGACCTTATGAGGGAACCCAGACCGCCAAGCTGAAAGTGACCAAGGGCGGACCCCTGCCTTTCGCCTGGGATATCCTGTCTCCCCAGTTTATGTACGGCAGCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACCTGAAGCTGAGCTTCCCTGAGGGCTTCAAGTGGGAGAGAGTGATGAATTTCGAGGACGGCGGAGTCGTGACAGTGACCCAGGATAGCTCTCTGCAGGACGGCGAGTTCATCTACAAAGTGAAGCTGCGGGGCACCAACTTCCCCAGCGACGGACCCGTGATGCAGAAAAAGACCATGGGCTGGGAGGCCAGCTCCGAGAGAATGTACCCAGAGGACGGGGCCCTGAAGGGGGAGATCAAGCAGCGGCTGAAACTGAAGGATGGCGGCCACTACGACGCAGAAGTGAAAACCACCTACAAGGCCAAGAAACCTGTGCAGCTGCCTGGCGCCTACAATGTGAACATCAAGCTGGACATTACCAGCCACAACGAGGACTACACCATCGTGGAACAGTACGAGCGGGCCGAGGGCAGGCATTCTACAGGCGGAATGGATGAACTGTATAAGTGCGTGAC CGACTA SEQ ID NO: 35MALPVTALLLPLALLLHAARPEQKLISEEDLDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCGSGSGSGSGSDRRGGRMLKHKRQRDDGEGRGEVGSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKRMEHLYSMKCKNVVPLFDLLLEMLDAHRLHAPTSGSGSGSGSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRSGSGSGSLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGSGSGSMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA EGRHSTGGMDELYKCVTD

Example 2—NHR-LBD Containing Anti-CD19 Chimeric Antigen ReceptorsExhibit Ligand-Induced Surface Expression of the CAR

Jurkat T cells were transduced with lentiviral vectors encoding thecanonical CAR or with anti-CD19 (FMC63) ERa-LBD containing the v2, v3,and v4 CAR constructs described in Example 1. The cells were then eitherleft untreated, or were treated with, a ligand for the ERa-LBD. Afterone hour of drug treatment, the cells were stained with Protein L-biotin(Protein L binds to the extracellular scFv), and then withstreptavidin-BV421 to detect surface expression of the CAR. The cellswere subsequently analyzed by flow cytometry for mCherry expression andfor the presence of CAR on the cell surface (FIG. 2A). Canonical CAR wasexpressed on the surface at identical levels regardless of the presenceor absence of the drug (FIG. 2A, dot plots under the heading “CanonicalCAR”). In contrast, ERa-LBD-containing v2 and v4 CARs (exemplarysingle-chain chimeric antigen receptors described herein) are expressedin the presence of the ligand (as assayed by mCherry-positiveexpression), but in the absence of the ligand, no CAR is detected on thecell surface (FIG. 2A, top row, Protein L staining, dot plots under theheadings “v2” and “v4”). After treatment with 500 ng/mL 4-OHT, CAR isdetected on cell surface (FIG. 2A, bottom row, Protein L staining) atlevels comparable to control CAR. Notably, not all designs behaveidentically: v3 has some basal surface expression of CAR in the absenceof ligand (FIG. 2A, top dot plot under the heading “v3”), and expressionis further increased upon ligand addition (FIG. 2A, bottom dot plotunder the heading “v3”).

Primary CD4 and CD8 human T cells were transduced with lentiviralvectors encoding the v4 CAR constructs. Cells were treated as describedin this Example, and were stained using an anti-cMyc antibody to detectthe cMyc-tagged scFv on the cell surface. Surface induction was observedin both CD4 and CD8 T cells after co-culture of the T cells with 500ng/mL 4-OHT ligand (FIG. 2B), demonstrating that the surface inductionphenotype extends from Jurkat to primary human T cells.

Example 3—NHR-LBD Fusion Proteins Exhibit Ligand-Induced SurfaceExpression in Diverse Cell Types

HEK-293T cells were transduced with lentiviral vectors encodingERa-LBD-containing FMC63 CAR fusions described in Example 1. The cellswere either left untreated, or were incubated with 500 ng/mL 4-OHT fortwo hours, and then stained for surface CAR expression. Consistent withERa LBD results in Jurkat cells and primary T cells demonstrated inExample 2, several ERa-LBD CAR fusions exhibited minimal surfaceexpression of CAR in the absence of ligand, and strongly up-regulatedsurface CAR upon ligand treatment (FIG. 3). These data indicate thatligand-induced surface expression of NHR-LBD CAR fusions operates indiverse cell types including immortalized (Jurkat and HEK-293T) andprimary cells (Human CD4 and CD8 T cells), as well as lymphoid (Jurkatand human T) and non-lymphoid (HEK-293T) cell types.

Example 4—Ligand-Induced Surface Expression of NHR LBD-CAR Fusions isConserved Between Different scFvs

To demonstrate the conserved function and modularity of NHR LBD-CARfusion proteins, the FMC63 scFv in V4 was replaced with a different scFv(J6M0) that recognizes B-cell maturation antigen (“BCMA”). Primary humanCD4 T cells were transduced with lentiviral vectors encoding the NHR-LBDCAR fusions, and were either left untreated, or were treated with 500ng/mL of the ERa ligand 4-OHT. Similar to the FMC63 NHR LBD-CAR fusions,the NHR LBD-CAR having the J6M0 scFv displayed ligand-induced surfaceexpression (FIG. 4).

Example 5—Ligand-Induced Surface Trafficking of NHR LBD Fusion Proteinsdoes not Require scFv or Intracellular Domains from the Canonical CAR

The LBD-CAR fusions tested in Example 2 contain a number of proteinmotifs in addition to the NHR LBD domain, including a CD137intracellular domain, a CD3z intracellular signaling domain, and an scFvdomain. To determine if the intracellular portions of the CAR constructs(the CD137 intracellular domain and CD3z intracellular domain) wereinvolved in ligand-induced surface trafficking, a construct lackingthese regions (“v5”, FIG. 5A, left) was generated and tested in primaryhuman T cells. Construct v5 includes the scFv domain and the mCherrydomain. Construct v5 exhibited ligand-induced trafficking comparable tothat of constructs v2 and v4 in Example 2, indicating the CARintracellular motifs were not responsible for the ligand-inducedtrafficking (FIG. 5A, right). The nucleotide (SEQ ID NO: 36) and aminoacid (SEQ ID NO: 37) sequence of v5 are shown below.

SEQ ID NO: 36 ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCCCTGCTGCTGCATGCCGCTAGACCTGAGCAGAAGCTGATCTCCGAAGAGGACCTGGACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGATAGAGTGACCATCAGCTGCAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAACCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCAGACTGCACAGCGGCGTGCCCAGCAGATTTTCTGGCAGCGGCTCCGGCACCGACTACAGCCTGACCATCTCCAACCTGGAACAGGAAGATATCGCTACCTACTTCTGTCAGCAAGGCAACACCCTGCCCTACACCTTCGGCGGAGGCACCAAGCTGGAAATCACAGGCGGCGGAGGATCTGGCGGAGGCGGAAGTGGCGGAGGGGGATCTGAAGTGAAACTGCAGGAAAGCGGCCCTGGCCTGGTGGCCCCATCTCAGTCTCTGAGCGTGACCTGTACCGTGTCCGGCGTGTCCCTGCCTGACTATGGCGTGTCCTGGATCAGACAGCCCCCCAGAAAGGGCCTGGAATGGCTGGGAGTGATCTGGGGCAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTGACCATCATCAAGGACAACTCCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTACGGCGGCAGCTACGCCATGGACTACTGGGGCCAGGGCACAAGCGTGACCGTGTCTAGCACAACCACCCCTGCCCCTAGACCTCCAACCCCAGCCCCTACAATCGCCAGCCAGCCTCTGTCTCTGAGGCCCGAGGCTTGTAGACCAGCTGCTGGCGGAGCCGTGCACACCAGAGGACTGGATTTCGCCTGCGACATCTACATCTGGGCCCCTCTGGCCGGCACATGTGGCGTGCTGCTGCTGAGCCTCGTGATCACCCTGTACTGCGGATCCGGCAGCGGATCTGGCAGTGGAAGCGATAGAAGAGGCGGCAGAATGCTGAAACACAAGCGGCAGAGGGACGACGGGGAAGGCAGAGGCGAAGTGGGATCTGCCGGCGATATGAGAGCCGCCAACCTGTGGCCTAGCCCCCTGATGATCAAGCGGAGCAAGAAGAACTCCCTGGCCCTGAGCCTGACCGCCGACCAGATGGTGTCTGCCCTGCTGGATGCCGAGCCCCCCATCCTGTACAGCGAGTACGACCCCACCAGACCCTTCAGCGAGGCCAGCATGATGGGCCTGCTGACCAACCTGGCCGACCGGGAACTGGTGCACATGATCAACTGGGCCAAGCGGGTGCCCGGCTTCGTGGATCTGACACTGCACGACCAGGTGCACCTGCTGGAATGCGCTTGGCTGGAAATCCTGATGATCGGCCTCGTGTGGCGGAGCATGGAACACCCTGGCAAGCTGCTGTTCGCCCCCAACCTGCTGCTGGACCGGAACCAGGGCAAATGCGTGGAAGGCATGGTGGAAATCTTCGACATGCTGCTGGCCACCTCCAGCCGGTTCCGGATGATGAACCTGCAGGGCGAAGAGTTCGTGTGTCTGAAGTCCATCATCCTGCTGAATAGCGGCGTGTACACCTTCCTGAGCAGCACCCTGAAAAGCCTGGAAGAAAAGGACCACATCCACCGGGTGCTGGACAAGATCACCGACACCCTGATTCACCTGATGGCCAAGGCCGGACTGACCCTGCAGCAGCAGCATCAGAGACTGGCTCAGCTGCTGCTGATCCTGTCCCACATCCGGCACATGAGCAACAAGCGGATGGAACATCTGTACAGCATGAAGTGCAAGAACGTGGTGCCTCTGTTCGATCTGCTGCTGGAAATGCTGGACGCCCACAGGCTGCACGCCCCAACATCCGGATCTGGCTCTGGAAGCGGCAGCATGGTGTCTAAGGGGGAAGAGGACAACATGGCCATCATCAAAGAATTCATGCGGTTCAAGGTGCACATGGAAGGCTCCGTGAATGGCCACGAATTCGAGATCGAGGGGGAGGGCGAGGGCAGACCTTATGAGGGAACCCAGACCGCCAAGCTGAAAGTGACCAAGGGCGGACCCCTGCCTTTCGCCTGGGATATCCTGTCTCCCCAGTTTATGTACGGCAGCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACCTGAAGCTGAGCTTCCCTGAGGGCTTCAAGTGGGAGAGAGTGATGAATTTCGAGGACGGCGGAGTCGTGACAGTGACCCAGGATAGCTCTCTGCAGGACGGCGAGTTCATCTACAAAGTGAAGCTGCGGGGCACCAACTTCCCCAGCGACGGACCCGTGATGCAGAAAAAGACCATGGGCTGGGAGGCCAGCTCCGAGAGAATGTACCCAGAGGACGGGGCCCTGAAGGGGGAGATCAAGCAGCGGCTGAAACTGAAGGATGGCGGCCACTACGACGCAGAAGTGAAAACCACCTACAAGGCCAAGAAACCTGTGCAGCTGCCTGGCGCCTACAATGTGAACATCAAGCTGGACATTACCAGCCACAACGAGGACTACACCATCGTGGAACAGTACGAGCGGGCCGAGGGCAGGCATTCTACAGGCGGAATGGATGAACTGTATAAGTGCGTGACCGACTAG SEQ ID NO: 37MALPVTALLLPLALLLHAARPEQKLISEEDLDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCGSGSGSGSGSDRRGGRMLKHKRQRDDGEGRGEVGSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKRMEHLYSMKCKNVVPLFDLLLEMLDAHRLHAPTSGSGSGSGSMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKCVTD

To test whether the extracellular scFv was involved in theligand-induced surface expression, constructs lacking the scFv weregenerated; in its place a FLAG tag was inserted to allow surfacedetection of the protein (nucleic acid and polypeptide sequences forthis construct are shown below as SEQ ID NOs: 38 and 39, respectively).Jurkat T cells were transduced with a lentiviral vector encoding theERa-LBD fusion protein, and were stained for surface expression of thepolypeptide in the presence and absence of the ERa ligand 4-OHT. Ligandtreatment induced robust surface detection of the polypeptide (FIG. 5B),indicating that NHR-LBD-mediated surface expression does not require anscFv, and that the technology is broadly generalizable to diversepolypeptides.

SEQ ID NO: 38 ATGATCCACCTGGGACACATCCTGTTTTTGCTGCTGCTGCCAGTGGCTGCCGCCgattacaaagacgacgatgataaaCAGACAACACCAGGCGAGAGATCTAGCCTGCCCGCCTTCTACCCTGGCACCAGCGGCTCTTGTTCTGGCTGTGGCAGCCTGTCTCTGCCCatctatatttgggcacccctggctggaacctgcggagtgctgctgctgtctctcgtgattacactgtattgcAAAAGGGGCCGGAAAAAGCTGCTGTATATTTTCAAACAGCCTTTTATGAGGCCTGTGCAGACAACACAGGAAGAGGACGGCTGTAGCTGTCGGTTCCCCGAAGAGGAAGAGGGGGGCTGCGAACTGggatcaggcagtggctctggcagcGATAGAAGAGGCGGCAGAATGCTGAAACACAAGCGGCAGAGGGACGACGGGGAAGGCAGAGGCGAAGTGGGATCTGCCGGCGATATGAGAGCCGCCAACCTGTGGCCTAGCCCCCTGATGATCAAGCGGAGCAAGAAGAACTCCCTGGCCCTGAGCCTGACCGCCGACCAGATGGTGTCTGCCCTGCTGGATGCCGAGCCCCCCATCCTGTACAGCGAGTACGACCCCACCAGACCCTTCAGCGAGGCCAGCATGATGGGCCTGCTGACCAACCTGGCCGACCGGGAACTGGTGCACATGATCAACTGGGCCAAGCGGGTGCCCGGCTTCGTGGATCTGACACTGCACGACCAGGTGCACCTGCTGGAATGCGCTTGGCTGGAAATCCTGATGATCGGCCTCGTGTGGCGGAGCATGGAACACCCTGGCAAGCTGCTGTTCGCCCCCAACCTGCTGCTGGACCGGAACCAGGGCAAATGCGTGGAAGGCATGGTGGAAATCTTCGACATGCTGCTGGCCACCTCCAGCCGGTTCCGGATGATGAACCTGCAGGGCGAAGAGTTCGTGTGTCTGAAGTCCATCATCCTGCTGAATAGCGGCGTGTACACCTTCCTGAGCAGCACCCTGAAAAGCCTGGAAGAAAAGGACCACATCCACCGGGTGCTGGACAAGATCACCGACACCCTGATTCACCTGATGGCCAAGGCCGGACTGACCCTGCAGCAGCAGCATCAGAGACTGGCTCAGCTGCTGCTGATCCTGTCCCACATCCGGCACATGAGCAACAAGCGGATGGAACATCTGTACAGCATGAAGTGCAAGAACGTGGTGCCTCTGTTCGATCTGCTGCTGGAAATGCTGGACGCCCACAGGCTGCACGCCCCAACATCCaGATCCggatctggaagtggctccCTGAGAGTGAAGTTTAGCAGAAGCGCCGACGCCCCTGCCTATCAGCAGGGACAGAACCAGCTGTATAACGAGCTGAACCTGGGCAGGCGGGAAGAGTACGACGTGCTGGATAAGAGGCGGGGCAGGGACCCTGAAATGGGCGGCAAACCCAGACGGAAGAACCCCCAGGAAGGCCTGTACAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGGCGGAGAGGCAAGGGACATGATGGCCTGTACCAGGGCCTGTCCACCGCCACCAAGGACACCTATGACGCCCTGCACATGCAGGCCCTGCCTCCAAGAggaagtggatctgggagcggctctatggtgtctaagggggaagaggacaacatggccatcatcaaagaattcatgcggttcaaggtgcacatggaaggctccgtgaatggccacgaattcgagatcgagggggagggcgagggcagaccttatgagggaacccagaccgccaagctgaaagtgaccaagggcggacccctgcctttcgcctgggatatcctgtctccccagtttatgtacggcagcaaggcctacgtgaagcaccccgccgacatccccgactacctgaagctgagcttccctgagggcttcaagtgggagagagtgatgaatttcgaggacggcggagtcgtgacagtgacccaggatagctctctgcaggacggcgagttcatctacaaagtgaagctgcggggcaccaacttccccagcgacggacccgtgatgcagaaaaagaccatgggctgggaggccagctccgagagaatgtacccagaggacggggccctgaagggggagatcaagcagcggctgaaactgaaggatggcggccactacgacgcagaagtgaaaaccacctacaaggccaagaaacctgtgcagctgcctggcgcctacaatgtgaacatcaagctggacattaccagccacaacgaggactacaccatcgtggaacagtacgagcgggccgagggcaggcattctacaggcggaatggatgaactgtataagtgcgtgaccgacTAG SEQ ID NO: 39MIHLGHILFLLLLPVAAADYKDDDDKQTTPGERSSLPAFYPGTSGSCSGCGSLSLPIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGSGSGSGSDRRGGRMLKHKRQRDDGEGRGEVGSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKRMEHLYSMKCKNVVPLFDLLLEMLDAHRLHAPTSRSGSGSGSLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGSGSGSMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKCVTD

The polypeptide of SEQ ID NO: 39 includes the following domains: Dap10signal peptide (MIHLGHILFLLLLPVAAA, SEQ ID NO: 40), flag tag (DYKDDDDK,SEQ ID NO: 41), Dap10 extracellular domain(QTTPGERSSLPAFYPGTSGSCSGCGSLSLP, SEQ ID NO: 42), CD8 transmembranedomain (IYIWAPLAGTCGVLLLSLVITLYC, SEQ ID NO: 43), 4-1BB costimulatorydomain (KRGRKKLLYIFKQPFMRPVQTTQ EEDGCSCRFPEEEEGGCEL, SEQ ID NO: 44),first linker (GSGSGSGS, SEQ ID NO: 45), modified ERa domain(DRRGGRMLKHKRQRDDGEGRGEVGSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKRMEHLYSMKCKNVVPLFDLLLEMLDAHRLHAPTS, SEQ ID NO: 46), secondlinker (RSGSGSGS, SEQ ID NO: 47), CD3 zeta signaling domain(LRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALH MQALPPR, SEQ IDNO: 48), third linker (GSGSGSGS, SEQ ID NO: 49), mCherry(MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKCVTD, SEQ ID NO: 50).

Example 6—Ligand-Induced Surface Expression of NHR-LBD Fusion ConstructsExtends to Different NHRs Including Progesterone Receptor (PR)

To test whether progesterone receptor (“PR”) LBD fusions exhibitedligand-induced surface expression, a construct (v33) was generated(nucleic acid and polypeptide sequences for this construct are shownbelow as SEQ ID NOs: 51 and 52, respectively). The tested constructcontained an N-terminal FLAG tag for surface detection. Jurkat T cellswere transduced with lentiviral vectors encoding the construct, and wereeither left untreated, or were treated with 500 ng/mL mifepristone, a PRligand. The cells were stained for surface protein expression after twohours of drug treatment. The construct exhibited ligand-induced surfaceexpression of the polypeptide as detected by flow cytometry (FIG. 6),indicating that other NHR-LBDs (aside from ERa) could be used togenerate constructs with ligand-inducible surface expression, and thatligand-induced surface expression of NHR-LBD fusions proteins is agenerizable phenomenon.

SEQ ID NO: 51 ATGATCCACCTGGGACACATCCTGTTTTTGCTGCTGCTGCCAGTGGCTGCCGCCgattacaaagacgacgatgataaaCAGACAACACCAGGCGAGAGATCTAGCCTGCCCGCCTTCTACCCTGGCACCAGCGGCTCTTGTTCTGGCTGTGGCAGCCTGTCTCTGCCCatctatatttgggcacccctggctggaacctgcggagtgctgctgctgtctctcgtgattacactgtattgcAAAAGGGGCCGGAAAAAGCTGCTGTATATTTTCAAACAGCCTTTTATGAGGCCTGTGCAGACAACACAGGAAGAGGACGGCTGTAGCTGTCGGTTCCCCGAAGAGGAAGAGGGGGGCTGCGAACTGggatcaggcagtggctctggcagcGGGCAAGACATTCAGCTCATACCTCCTTTGATAAATTTGCTGATGTCTATAGAACCAGATGTCATATACGCTGGTCACGACAATACGAAACCGGACACATCTTCATCTTTGCTTACCTCTCTGAATCAACTGGGTGAACGACAGCTCCTGAGTGTTGTTAAGTGGTCTAAAAGCCTCCCGGGCTTCAGGAATTTGCACATAGACGACCAAATCACGCTCATCCAATATTCCTGGATGAGTCTCATGGTCTTTGGTCTCGGTTGGCGCAGCTATAAGCACGTCTCTGGCCAGATGTTGTATTTCGCACCAGACCTGATCCTGAACGAACAGAGGATGAAGGAATCAAGCTTTTACTCTCTCTGCTTGACTATGTGGCAAATCCCCCAAGAATTCGTGAAACTTCAAGTTTCCCAAGAAGAATTCCTCTGCATGAAAGTCCTTCTTTTGCTCAACACGATTCCCCTGGAAGGCTTGAGGTCTCAAACGCAATTCGAGGAGATGCGGAGTAGCTATATACGCGAACTCATCAAGGCCATCGGTTTGCGGCAAAAGGGAGTGGTCTCTAGTAGCCAACGATTTTACCAGCTGACTAAGCTCCTTGACAACCTTCACGATCTCGTCAAACAACTGCACCTGTACTGTCTTAACACATTTATACAATCACGGGCACTTTCTGTAGAGTTCCCAGAGATGATGTCTGAGGTCATCGCAGCCCAACTTCCGAAAATTCTTGCAGGAATGGTGAAGCCACTTCTGTTCCATAAGAAAaGATCCggatctggaagtggctccCTGAGAGTGAAGTTTAGCAGAAGCGCCGACGCCCCTGCCTATCAGCAGGGACAGAACCAGCTGTATAACGAGCTGAACCTGGGCAGGCGGGAAGAGTACGACGTGCTGGATAAGAGGCGGGGCAGGGACCCTGAAATGGGCGGCAAACCCAGACGGAAGAACCCCCAGGAAGGCCTGTACAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGGCGGAGAGGCAAGGGACATGATGGCCTGTACCAGGGCCTGTCCACCGCCACCAAGGACACCTATGACGCCCTGCACATGCAGGCCCTGCCTCCAAGAggaagtggatctgggagcggctctatggtgtctaagggggaagaggacaacatggccatcatcaaagaattcatgcggttcaaggtgcacatggaaggctccgtgaatggccacgaattcgagatcgagggggagggcgagggcagaccttatgagggaacccagaccgccaagctgaaagtgaccaagggcggacccctgcctttcgcctgggatatcctgtctccccagtttatgtacggcagcaaggcctacgtgaagcaccccgccgacatccccgactacctgaagctgagcttccctgagggcttcaagtgggagagagtgatgaatttcgaggacggcggagtcgtgacagtgacccaggatagctctctgcaggacggcgagttcatctacaaagtgaagctgcggggcaccaacttccccagcgacggacccgtgatgcagaaaaagaccatgggctgggaggccagctccgagagaatgtacccagaggacggggccctgaagggggagatcaagcagcggctgaaactgaaggatggcggccactacgacgcagaagtgaaaaccacctacaaggccaagaaacctgtgcagctgcctggcgcctacaatgtgaacatcaagctggacattaccagccacaacgaggactacaccatcgtggaacagtacgagcgggccgagggcaggcattctacaggcggaatggatgaactgtat aagtgcgtgaccgac SEQID NO: 52 MIHLGHILFLLLLPVAAADYKDDDDKQTTPGERSSLPAFYPGTSGSCSGCGSLSLPIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGSGSGSGSGQDIQLIPPLINLLMSIEPDVIYAGHDNTKPDTSSSLLTSLNQLGERQLLSVVKWSKSLPGFRNLHIDDQITLIQYSWMSLMVFGLGWRSYKHVSGQMLYFAPDLILNEQRMKESSFYSLCLTMWQIPQEFVKLQVSQEEFLCMKVLLLLNTIPLEGLRSQTQFEEMRSSYIRELIKAIGLRQKGVVSSSQRFYQLTKLLDNLHDLVKQLHLYCLNTFIQSRALSVEFPEMMSEVIAAQLPKILAGMVKPLLFHKKRSGSGSGSLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGSGSGSMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELY KCVTD

The polypeptide of SEQ ID NO: 52 includes the following domains: Dap10signal peptide (MIHLGHILFLLLLPVAAA, SEQ ID NO: 53), flag tag (DYKDDDDK,SEQ ID NO: 54), Dap10 extracellular domain(QTTPGERSSLPAFYPGTSGSCSGCGSLSLP, SEQ ID NO: 55), CD8 transmembranedomain (IYIWAPLAGTCGVLLLSLVITLYC, SEQ ID NO: 56), 4-1BB costimulatorydomain (KRGRKKLLYIFKQPFMRPVQTTQ EEDGCSCRFPEEEEGGCEL, SEQ ID NO: 57),first linker (GSGSGSGS, SEQ ID NO: 58), Progesterone receptor LBD(GQDIQLIPPLINLLMSIEPDVIYAGHDNTKPDTSSSLLTSLNQLGERQLLSVVKWSKSLPGFRNLHIDDQITLIQYSWMSLMVFGLGWRSYKHVSGQMLYFAPDLILNEQRMKESSFYSLCLTMWQIPQEFVKLQVSQEEFLCMKVLLLLNTIPLEGLRSQTQFEEMRSSYIRELIKAIGLRQKGVVSSSQRFYQLTKLLDNLHDLVKQLHLYCLNTFIQSRALSVEFPEMMSEVIAAQLPKILAGMVKPLL FHKK, SEQ ID NO:59), second linker (RSGSGSGS, SEQ ID NO: 60), CD3zeta signaling domain(LRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR, SEQ ID NO: 61), third linker (GSGSGSGS, SEQ IDNO: 62), and mCherry (MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKCVTD, SEQ ID NO: 63).

The nucleotide sequence of SEQ ID NO: 51 includes the followingsequences encoding the indicated domains: Dap10 signal peptide(ATGATCCACCTGGGACACAT CCTGTTTTTGCTGCTGCTGCCAGTGGCTGCCGCC, SEQ ID NO:63), flag tag (gattacaaagacgacgatgataaa, SEQ ID NO: 64), Dap10extracellular domain (CAGACAACACCAGGCGAGAGATCTAGCCTGCCCGCCTTCTACCCTGGCACCAGCGGCTCTTGTTCTGGCTGTGGCAGCCTGTCTCTGCCC, SEQ ID NO: 65), CD8 transmembrane domain(atctatatttgggcaccectggctggaacctgeggagtgctgctgctgtctctcgtgattacactgtattgc,SEQ ID NO: 66), 4-1BB costimulatory domain (AAAAGGGGCCGGAAAAAGCTGCTGTATATTTTCAAACAGCCTTTTATGAGGCCTGTGCAGACAACACAGGAAGAGGACGGCTGTAGCTGTCGGTTCCCCGAAGAGGAAGAGGGGGGCTGCGAACTQ SEQ ID NO: 67), firstlinker (ggatcaggcagtggctctggcagc, SEQ ID NO: 68), Progesterone receptorLBD (GGGCAAGACATTCAGCTCATACCTCCTTTGATAAATTTGCTGATGTCTATAGAACCAGATGTCATATACGCTGGTCACGACAATACGAAACCGGACACATCTTCATCTTTGCTTACCTCTCTGAATCAACTGGGTGAACGACAGCTCCTGAGTGTTGTTAAGTGGTCTAAAAGCCTCCCGGGCTTCAGGAATTTGCACATAGACGACCAAATCACGCTCATCCAATATTCCTGGATGAGTCTCATGGTCTTTGGTCTCGGTTGGCGCAGCTATAAGCACGTCTCTGGCCAGATGTTGTATTTCGCACCAGACCTGATCCTGAACGAACAGAGGATGAAGGAATCAAGCTTTTACTCTCTCTGCTTGACTATGTGGCAAATCCCCCAAGAATTCGTGAAACTTCAAGTTTCCCAAGAAGAATTCCTCTGCATGAAAGTCCTTCTTTTGCTCAACACGATTCCCCTGGAAGGCTTGAGGTCTCAAACGCAATTCGAGGAGATGCGGAGTAGCTATATACGCGAACTCATCAAGGCCATCGGTTTGCGGCAAAAGGGAGTGGTCTCTAGTAGCCAACGATTTTACCAGCTGACTAAGCTCCTTGACAACCTTCACGATCTCGTCAAACAACTGCACCTGTACTGTCTTAACACATTTATACAATCACGGGCACTTTCTGTAGAGTTCCCAGAGATGATGTCTGAGGTCATCGCAGCCCAACTTCCGAAAATTCTTGCAGGAATGGTGAAGCCACTTCTGTTCCATAAGAAA, SEQ ID NO: 69), second linker(ggatctggaagtggctcc, SEQ ID NO: 70), CD3zeta signaling domain(CTGAGAGTGAA GTTTAGCAGAAGCGCCGACGCCCCTGCCTATCAGCAGGGACAGAACCAGCTGTATAACGAGCTGAACCTGGGCAGGCGGGAAGAGTACGACGTGCTGGATAAGAGGCGGGGCAGGGACCCTGAAATGGGCGGCAAACCCAGACGGAAGAACCCCCAGGAAGGCCTGTACAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGGCGGAGAGGCAAGGGACATGATGGCCTGTACCAGGGCCTGTCCACCGCCACCAAGGACACCTATGACGCCCTGCACATGCAGGCCCTGCCTCCAAGA, SEQ ID NO: 71), third linker(ggaagtggatctgggagcggctct, SEQ ID NO: 72), and mCherry(atggtgtctaagggggaagaggacaacatggccatcatcaaagaattcatgcggttcaaggtgcacatggaaggctccgtgaatggccacgaattcgagatcgagggggagggcgagggcagaccttatgagggaacccagaccgccaagctgaaagtgaccaagggcggacccctgcctttcgcctgggatatcctgtctccccagtttatgtacggcagcaaggcctacgtgaagcaccccgccgacatccccgactacctgaagctgagatccctgagggcttcaagtgggagagagtgatgaatttcgaggacggcggagtcgtgacagtgacccaggatagctctctgcaggacggcgagttcatctacaaagtgaagctgcggggcaccaacttccccagcgacggacccgtgatgcagaaaaagaccatgggctgggaggccagctccgagagaatgtacccagaggacggggccctgaagggggagatcaagcagcggctgaaactgaaggatggcggccactacgacgcagaagtgaaaaccacctacaaggccaagaaacctgtgcagctgcctggcgcctacaatgtgaacatcaagctggacattaccagccacaacgaggactacaccatcgtggaacagtacgagcgggccgagggcaggcattctacaggcggaatggatgaactgtataagtgcgtgaccgac, SEQ ID NO: 73).

Example 7—Ligand-Induced NHR-LBD CAR T Cell Activation is Antigen- andDrug-Dependent

Primary human T cells were transduced with lentiviral vectors encodingJ6M0-ERa-LBD CAR construct. T cell activation was assessed by surfacestaining for the canonical T cell activation marker, CD69, afterovernight co-culture with antigen-negative (K562 cell line) andantigen-positive (MM1S cell line) targets in the presence of increasingconcentrations of 4-OHT (FIG. 7A). A unique dose-dependent T cellresponse was observed for each construct, with the proximity of the LBDto the cell membrane correlating to background activation (activation inthe absence of ligand) and sensitivity to ligand. All constructsdisplayed activation levels comparable to the positive control(canonical CAR) at increasing drug concentrations.

The ability of the J6M0-ERa-LBD CAR cells to produce inflammatorycytokines was assessed via IFN-γ ELISA on co-culture supernatantsisolated from overnight co-cultures of T cells with antigen-negative(K562 cell line) and antigen-positive (MM1S cell line) targets in thepresence of increasing concentrations of 4-OHT. IFN-γ production wasshown to be dose-dependent, with all NHR-LBD constructs producingcytokine at increasing ligand concentrations (FIG. 7B).

Example 8—Ligand-Induced NHR-LBD CAR T Cell Proliferation and Killingare Antigen- and Ligand-Dependent

Primary human CD4 T cells were transduced with lentiviral vectorsencoding V4 FMC63-ERa-LBD CAR or canonical CAR controls. The cells werelabeled with carboxyfluorescein succinimidyl ester (“CFSE”) prior toco-culture with either antigen-negative (K562 cell line) orantigen-positive (Raji cell line) targets. The cells were alsoco-cultured with increasing concentrations of ligand (4-OHT). Three daysafter co-culture, cells were analyzed via flow cytometry andproliferation was assessed by dilution of CFSE dye on CD4+ T cells.FMC63-ERa-LBD CART cells proliferated only in the presence of antigenand ligand (FIG. 8A).

Primary human T cells were transduced with lentiviral vectors encodingthe J6M0-ERa-LBD CAR construct described in Example 7. The ability of Tcells bearing the J6M0-ERa-LBD CAR construct to kill antigen-positivetargets (MM1S cell line) in a drug-dependent manner was assessed via aflow cytometry-based killing assay. Target cells were labeled with CFSEprior to co-culture, and the number of cells remaining in each wellafter co-culture was determined via quantification of a fixed volumeusing the Intellicyt flow cytometer. Specific lysis was determined bycomparing the number of targets remaining per experimental well to thenumber of targets remaining in control wells that contained both targetsand non-antigen specific conventional CAR T cells (−CTRL). Specificlysis was reported as a percentage derived from the following equation:1−(# events per experiment well/# average events−CTRL wells). Targetcell killing was dependent on the dose of the ligand, with allconstructs eliminating antigen-positive targets at levels comparable tothe conventional CAR (+CTRL) at higher drug doses (FIG. 8B).

Example 9—The Location of the LBD within the CAR Polypeptide DeterminesSensitivity to Ligand-Induced Surface Expression of NHR-LBD CAR

The dose responsiveness of the V2, V3, and V4 constructs shown in FIG. 1was compared in Jurkat T cells. Detection of surface expression ofNHR-LBD CAR protein on Jurkat T cells was performed after incubation forone hour with 4-OHT ligand at the concentrations indicated on the x-axisof FIG. 9. Surface receptor expression was detected using an anti-cMycantibody, and was reported as a percentage of reporter-positive cells(as determined by mCherry expression). The ligand dose at which surfaceexpression was detected for each construct corresponded to the locationof the LBD, in relation to the surface membrane, for each construct.Construct V4, which harbors the LBD immediately adjacent to the surfacemembrane, showed no detectable surface expression in the absence ofligand, with surface expression detected only at higher concentrationsof 4-OHT. In contrast, when the LBD was located further from themembrane (see e.g., V2), a higher basal level of surface expression wasdetected (−20%), and enhanced sensitivity to 4-OHT was observed.Activation of Jurkat T cells with PMA/Ionomycin did not lead toligand-independent NHR-LBD CAR surface expression, demonstrating thatactivation of the T cell alone (through the endogenous TCR, for example)does not result in trafficking of the receptor to the cell surface.

Example 10—Duration of NHR-LBD CAR Surface Expression is Dependent onthe Location of LBD in Polypeptide

T cells were transduced with FMC63-ERa-LBD CARs and were exposed to4-OHT at 35 ng/mL for one hour before either being stained for surfaceexpression using an anti-cMyc antibody, or being washed and culturedovernight in the absence of drug (timeline shown in FIG. 10A).

The number of cells expressing surface protein was reported as apercentage of reporter-positive cells (as determined by mCherryexpression). The duration of NHR-LBD CAR expression on the cell surfacefor constructs V2, V3, and V4 was determined. The construct thatharbored the LBD most proximal to the membrane (V4) showed the mostrapid and complete return to baseline 24 hours after ligand removal(FIG. 10B, bars labeled “V4”).

Example 11—Kinetics of Surface Expression in NHR-LBD-CAR Constructsafter Ligand Addition

Jurkat T cells were transduced with the FLAG-ERa-LBD construct used inExample 5 were left untreated or were treated with 500 ng/mL 4-OHT for15 minutes or 120 minutes, and surface expression was detected afterligand treatment. Ligand treatment induced surface expression within 15minutes, and further upregulation was observed over time (FIG. 11A).

Human T cells were transduced with lentiviral vector encoding theFMC63-ERa-LBD CAR (v2 as shown in FIG. 1). The cells were treated withthe ERa ligand 4-OHT for the indicated duration, and then stained forsurface expression of the CAR (FIG. 11B). The data in FIG. 11B showsimilar kinetics for the ligand treatment induced surface expression aswas observed for Jurkat T cells.

Example 12—Brefeldin a Treatment Blocks Ligand-Induced SurfaceTrafficking of NHR LBD Fusions

Primary human T cells expressing FMC63-CAR with ERa LBD fusion were leftuntreated or were treated with 500 ng/mL 4-OHT ligand for two hours toinduce surface expression of the CAR. Prior to 4-OHT treatment, half thecells were pre-treated with 1 uM Brefeldin A (FIG. 12, bottom twopanels) to inhibit protein export from the Golgi. Brefeldin Apre-treatment prevented 4-OHT-induced increase in surface proteinexpression. Without wishing to be bound by theory, these data suggestthat increased surface expression arises from relocalization of a poolof polypeptides that is sequestered intracellularly in the absence ofligand.

Example 13—Generation of Constructs

A conventional chimeric antigen receptor (“Conventional CAR”, FIG. 13,left) was modified to include the estrogen receptor alpha (“ERa”) ligandbinding domain (“ERa LBD,” FIG. 1, in green) either in the intracellularportion of the protein (v2 CAR as described in Example 1) or in theextracellular portion of the protein (“Concealed Carry’ CAR ECD.v1” or“LBD-ECD CAR”). Point mutations were introduced into the ERa LBD domainof both v2 CAR and ECD.v1 CAR. The nucleic acid and polypeptidesequences of v2 CAR (are shown above as SEQ ID NOs: 30 and 31) andECD.v1 (SEQ ID NOs: 97 and 98) are provided below.

(ECD v.1 CAR) SEQ ID NO: 97atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgattcctgaacagaagctgataagtgaggaggacttggacatccagatgacccagaccaccagcagcctgagcgccagcctgggcgatagagtgaccatcagctgcagagccagccaggacatcagcaagtacctgaactggtatcagcagaaacccgacggcaccgtgaagctgctgatctaccacaccagcagactgcacagcggcgtgcccagcagattttctggcagcggctccggcaccgactacagcctgaccatctccaacctggaacaggaagatatcgctacctacttctgtcagcaaggcaacaccctgccctacaccttcggcggaggcaccaagctggaaatcacaggcggcggaggatctggcggaggcggaagtggcggagggggatctgaagtgaaactgcaggaaagcggccctggcctggtggccccatctcagtctctgagcgtgacctgtaccgtgtccggcgtgtccctgcctgactatggcgtgtcctggatcagacagccccccagaaagggcctggaatggctgggagtgatctggggcagcgagacaacctactacaacagcgccctgaagtcccggctgaccatcatcaaggacaactccaagagccaggtgttcctgaagatgaacagcctgcagaccgacgacaccgccatctactactgcgccaagcactactactacggcggcagctacgccatggactactggggccagggcacaagcgtgaccgtgtctagcggatccgatagaagaggcggcagaatgctgaaacacaagcggcagagggacgacggggaaggcagaggcgaagtgggatctgccggcgatatgagagccgccaacctgtggcctagccccctgatgatcaagcggagcaagaagaactccctggccctgagcctgaccgccgaccagatggtgtctgccctgctggatgccgagccccccatcctgtacagcgagtacgaccccaccagacccttcagcgaggccagcatgatgggcctgctgaccaacctggccgaccgggaactggtgcacatgatcaactgggccaagcgggtgcccggcttcgtggatctgacactgcacgaccaggtgcacctgctggaatgcgcttggctggaaatcctgatgatcggcctcgtgtggcggagcatggaacaccctggcaagctgctgttcgcccccaacctgctgctggaccggaaccagggcaaatgcgtggaaggcatggtggaaatcttcgacatgctgctggccacctccagccggttccggatgatgaacctgcagggcgaagagttcgtgtgtctgaagtccatcatcctgctgaatagcggcgtgtacaccttcctgagcagcaccctgaaaagcctggaagaaaaggaccacatccaccgggtgctggacaagatcaccgacaccctgattcacctgatggccaaggccggactgaccctgcagcagcagcatcagagactggctcagctgctgctgatcctgtcccacatccggcacatgagcaacaagcggatggaacatctgtacagcatgaagtgcaagaacgtggtgcctctgttcgatctgctgctggaaatgctggacgcccacaggctgcacgccccaacatccggatccaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctag (ECD v.1 CAR) SEQ ID NO:98 MLLLVTSLLLCELPHPAFLLIPEQKLISEEDLDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSGSDRRGGRMLKHKRQRDDGEGRGEVGSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKRMEHLYSMKCKNVVPLFDLLLEMLDAHRLHAPTSGSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRThe protein and nucleic acid sequence of each domain in ECD.v1, goingfrom the N-terminus to the C-terminus, in ECD.v1 are shown below.

(CSF2RA signal peptide) SEQ ID NO: 99atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagc attcctcctgattcct(CSF2RA signal peptide) SEQ ID NO: 100 MLLLVTSLLLCELPHPAFLLIP (cMyc tag)SEQ ID NO: 101 gaacagaagctgataagtgaggaggacttg (cMyc tag) SEQ ID NO: 102EQKLISEEDL (FMC63 V_(L)) SEQ ID NO: 103gacatccagatgacccagaccaccagcagcctgagcgccagcctgggcgatagagtgaccatcagctgcagagccagccaggacatcagcaagtacctgaactggtatcagcagaaacccgacggcaccgtgaagctgctgatctaccacaccagcagactgcacagcggcgtgcccagcagattttctggcagcggctccggcaccgactacagcctgaccatctccaacctggaacaggaagatatcgctacctacttctgtcagcaaggcaacaccctgccctacaccttcggcgga ggcaccaagctggaaatcaca(FMC63 V_(H)) SEQ ID NO: 104DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGG GTKLEIT ((G₄S)₃linker between V_(L) and V_(H)) SEQ ID NO: 105ggcggcggaggatctggcggaggcggaagtggcggagggggatct ((G₄S)₃ linker betweenV_(L) and V_(H)) SEQ ID NO: 106 GGGGSGGGGSGGGGS (FMC63 V_(H)) SEQ ID NO:107 gaagtgaaactgcaggaaagcggccctggcctggtggccccatctcagtctctgagcgtgacctgtaccgtgtccggcgtgtccctgcctgactatggcgtgtcctggatcagacagccccccagaaagggcctggaatggctgggagtgatctggggcagcgagacaacctactacaacagcgccctgaagtcccggctgaccatcatcaaggacaactccaagagccaggtgttcctgaagatgaacagcctgcagaccgacgacaccgccatctactactgcgccaagcactactactacggcggcagctacgccatggactactggggccagggcacaagcgtgac cgtgtctagc (FMC63V_(H)) SEQ ID NO: 108 EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYY YGGSYAMDYWGQGTSVTVSSGS Linker Between V_(H) Domain and Estrogen Receptor Alpha LigandBinding DomainggatccGs Linker Between VII Domain and Estrogen Receptor Alpha Ligand BindingDomainGS

(Estrogen receptor alpha ligand binding domain) (SEQuence encoding hingeunderlined, sequence encoding helix 12 in bold) SEQ ID NO: 109gatagaagaggcggcagaatgctgaaacacaagcggcagagggacgacggggaaggcagaggcgaagtgggatctgccggcgatatgagagccgccaacctgtggcctagccccctgatgatcaagcggagcaagaagaactccctggccctgagcctgaccgccgaccagatggtgtctgccctgctggatgccgagccccccatcctgtacagcgagtacgaccccaccagacccttcagcgaggccagcatgatgggcctgctgaccaacctggccgaccgggaactggtgcacatgatcaactgggccaagcgggtgcccggcttcgtggatctgacactgcacgaccaggtgcacctgctggaatgcgcttggctggaaatcctgatgatcggcctcgtgtggcggagcatggaacaccctggcaagctgctgttcgcccccaacctgctgctggaccggaaccagggcaaatgcgtggaaggcatggtggaaatcttcgacatgctgctggccacctccagccggttccggatgatgaacctgcagggcgaagagttcgtgtgtctgaagtccatcatcctgctgaatagcggcgtgtacaccttcctgagcagcaccctgaaaagcctggaagaaaaggaccacatccaccgggtgctggacaagatcaccgacaccctgattcacctgatggccaaggccggactgaccctgcagcagcagcatcagagactggctcagctgctgctgatcctgtcccacatccggcacatgagcaacaagcggatggaacatctgtacagcatgaagtgcaagaacgtggtgcctctgttcgatctgctgctggaaatgctggacgcccacaggctgcacgccccaacatcc (Estrogen receptor alphaligand binding domain) (hinge underlined, helix 12 in bold) SEQ ID NO:110 DRRGGRMLKHKRQRDDGEGRGEVGSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKRMEHLYSMKCKNVVPLFDLLLEMLDAHRLHAPTSGS-Linker Between Estrogen Receptor Alpha Ligand Binding Domain and CD8Alpha Hinge RegionggatccGS-Linker Between Estrogen Receptor Alpha Ligand Binding Domain and CD8Alpha Hinge RegionGS

(CD8 alpha hinge) SEQ ID NO: 111accacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccct ttactgc (CD8 alphahinge) SEQ ID NO: 112 TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAC (CD8alpha transmembrane domain) SEQ ID NO: 113gatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgc (CD8 alpha transmembrane domain) SEQ ID NO:114 DIYIWAPLAGTCGVLLLSLVITLYC (4-1BB costimulatory domain) SEQ ID NO:115 aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaa (4-1BB costimulatory domain) SEQ ID NO: 116KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE (CD3 zeta signaling domain)SEQ ID NO: 117 ctgagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc (CD3 zeta signaling domain) SEQID NO: 118 LRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKD TYDALHMQALPPR

Example 14—LBD-ECD Reduces Background Localization of the CAR to theCell Surface in the Absence of Ligand

Primary human T cells were transduced with lentiviral vectors encodingthe CC.v2 or LBD ECD.v1 construct as indicated. Cells were leftun-treated or treated with 500 ng/mL of 4-OHT. Duration of treatment wasas shown in FIG. 14. Cells were subsequently stained for surfaceexpression of the chimeric receptor using anti-Myc AlexaFluor 647antibody.

Placement of the LBD to the extracellular portion of the CAR reduces thebackground levels of CAR surface expression when the ligand has not beenadded, relative to when the LBD is placed in the intracellular portionof the CAR (FIG. 14). Both v2 CAR and ECD.v1 were expressed on thesurface of cells when 500 ng/ml of 4-OHT was added.

Example 15—LBD-ECD CAR Kills Cells in a Drug Dependent Manner

Primary human T cells were transduced with lentiviral vectors encodingthe LBD ECD.v1 or control CAR construct as indicated. T cells wereallowed to rest after expansion and subsequently co-cultured withluciferase expressing Nalm6 tumor cells at an effector:target ratioshown in FIG. 15. After 24 hours of co-culture the cells were lysed andluciferase substrate added to quantify the number of surviving tumorcells.

LBD ECD.v1 CAR kills cells in a drug dependent manner. The addition ofincreasing amounts of 4-OHT leads to greater levels of cell lysis(approximately 50% lysis at 500 ng/mL 4-OHT) (FIG. 15).

Example 16—LBD-ECD CAR is Localized Intracellularly in Activated T Cells

Primary human T cells were transduced with lentiviral vectors encodingthe LBD_ECD, CC.v2, or constitutive control CAR construct as indicated.After cells had rested, cells were re-stimulated with aCD3/aCD28dynabeads for 48 hours. Cells were subsequently stained with anti-Mycantibody to detect surface expression of the CAR, and analyzed via flowcytometry.

CARs with an extracellular LBD (LBD ECD v1) are localizedintracellularly in T cells that are activated when exposed to a CD3/CD28beads. CARs with an intracellular LBD (v2 CAR) show some localization tothe cell surface in activated T cells (FIG. 16).

Example 17—ECD.v1 CAR (LBD_ECD) Produces Cytokine IL-2

IL-2 production from the ECD.v1 CAR T cells was compared to theconventional CAR+ control after overnight co-culture of CAR-bearing Tcells with NALM-6 targets in the presence (500 ng/mL) or absence of4-hydroxytamoxifen (FIG. 16). IL-2 production from the ECD.v1 CARreceptor (FIG. 13) was comparable to the conventional CAR control,dependent upon 4-hydroxytamoxifen, and undetectable in the absence ofdrug.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims

What is claimed is:
 1. A single-chain chimeric polypeptide, wherein thesingle-chain chimeric polypeptide comprises: a transmembrane domain; anda hormone receptor ligand binding domain, wherein: the transmembranedomain and the hormone receptor ligand binding domain directly abut eachother or are separated by 1 to 500 amino acids; the transmembrane domainand the hormone receptor ligand binding domain are not both present inthe same endogenous single-chain polypeptide in a mammal; and whereinthe hormone receptor ligand binding domain is a ligand binding domain ofa steroid receptor.
 2. A nucleic acid comprising a nucleotide sequenceencoding the single-chain chimeric polypeptide of any claim
 1. 3. Avector comprising the nucleic acid of claim
 2. 4. An isolated cellcomprising the vector of claim
 3. 5. The single-chain chimericpolypeptide of claim 1, wherein the transmembrane domain is a domainfrom: a chain of a T cell receptor, β chain of a T cell receptor, ζchain of a T cell receptor, CD28, CD3ε, CD3δ, CD3γ, CD33, CD37, CD64,CD80, CD45, CD4, CD5, CD8a, CD9, CD16, CD22, CD86, CD134, CD137, orCD154.
 6. The single-chain chimeric polypeptide of claim 1, wherein thehormone receptor ligand binding domain is a ligand binding domain froman estrogen receptor, a progesterone receptor, or an androgen receptor.7. The single-chain chimeric polypeptide of claim 6, wherein the hormonereceptor ligand binding domain is a ligand binding domain from anestrogen receptor.
 8. The single-chain chimeric polypeptide of claim 1,wherein the transmembrane domain and the hormone receptor ligand bindingdomain directly abut each other.
 9. The single-chain chimericpolypeptide of claim 1, wherein the transmembrane domain and the hormonereceptor ligand binding domain are separated by 1 to 500 amino acids.10. The single-chain chimeric polypeptide of claim 9, wherein thetransmembrane domain and the hormone receptor ligand binding domain areseparated by 1 to 250 amino acids.
 11. The single-chain chimericpolypeptide of claim 10, wherein the transmembrane domain and thehormone receptor ligand binding domain are separated by 1 to 50 aminoacids.
 12. A single-chain chimeric polypeptide, wherein the single-chainchimeric polypeptide comprises: an extracellular hormone receptor ligandbinding domain; and a transmembrane domain, wherein: the extracellularhormone receptor ligand binding domain and the transmembrane domaindirectly abut each other or are separated by 1 to 700 amino acids; thetransmembrane domain and the extracellular hormone receptor ligandbinding domain are not both present in a single endogenous single-chainpolypeptide in a mammal; and wherein the extracellular hormone receptorligand binding domain is a ligand binding domain of a steroid receptor.13. A nucleic acid comprising a nucleotide sequence encoding thesingle-chain chimeric polypeptide of claim
 12. 14. A vector comprisingthe nucleic acid of claim
 13. 15. An isolated cell comprising the vectorof claim
 14. 16. The single-chain chimeric polypeptide of claim 12,wherein the transmembrane domain is a domain from: a chain of a T cellreceptor, β chain of a T cell receptor, ζ chain of a T cell receptor,CD28, CD3ε, CD3δ, CD3γ, CD33, CD37, CD64, CD80, CD45, CD4, CD5, CD8α,CD9, CD16, CD22, CD86, CD134, CD137, or CD154.
 17. The single-chainchimeric polypeptide of claim 12, wherein the extracellular hormonereceptor ligand binding domain is a ligand binding domain from anestrogen receptor, a progesterone receptor, or an androgen receptor. 18.The single-chain chimeric polypeptide of claim 17, wherein theextracellular hormone receptor ligand binding domain is a ligand bindingdomain from an estrogen receptor.
 19. The single-chain chimericpolypeptide of claim 12, wherein the extracellular hormone receptorligand binding domain is a ligand binding domain from an estrogenreceptor.
 20. The single-chain chimeric polypeptide of claim 19, whereinthe extracellular hormone receptor ligand binding domain is a ligandbinding domain from a human estrogen receptor.
 21. The single-chainchimeric polypeptide of claim 20, wherein the ligand binding domain ofthe human estrogen receptor has a wildtype sequence, except that theligand binding domain comprises one or both of a substitution at aminoacid position 521 and a substitution at amino acid position 537, eachnumbered relative to the full-length wildtype sequence of human hormonereceptor.
 22. The single-chain chimeric polypeptide of claim 12, whereinthe extracellular hormone receptor ligand binding domain and thetransmembrane domain directly abut each other.
 23. The single-chainchimeric polypeptide of claim 22, wherein the extracellular hormonereceptor ligand binding domain and the transmembrane domain areseparated by 1 to 700 amino acids.
 24. The single-chain chimericpolypeptide of claim 23, wherein the extracellular hormone receptorligand binding domain and the transmembrane domain are separated by 1 to500 amino acids.
 25. The single-chain chimeric polypeptide of claim 24,wherein the extracellular hormone receptor ligand binding domain and thetransmembrane domain are separated by 1 to 250 amino acids.
 26. Thesingle-chain chimeric polypeptide of claim 25, wherein the extracellularhormone receptor ligand binding domain and the transmembrane domain areseparated by 1 to 100 amino acids.
 27. The single-chain chimericpolypeptide of claim 26, wherein the extracellular hormone receptorligand binding domain and the transmembrane domain are separated by 1 to50 amino acids.
 28. The single-chain chimeric polypeptide of claim 27,wherein the extracellular hormone receptor ligand binding domain and thetransmembrane domain are separated by 1 to 25 amino acids.
 29. Thesingle-chain chimeric polypeptide of claim 12, wherein, when going inthe N-terminal to C-terminal direction, the single-chain chimericpolypeptide comprises an extracellular antigen-binding domain, theextracellular hormone receptor ligand binding domain, and thetransmembrane domain.
 30. The single-chain chimeric polypeptide of claim12, wherein, when going in the C-terminal to N-terminal direction, thesingle-chain chimeric polypeptide comprises an extracellularantigen-binding domain, the extracellular hormone receptor ligandbinding domain, and the transmembrane domain.
 31. The single-chainchimeric polypeptide of claim 12, wherein, when going in the N-terminalto C-terminal direction, the single-chain chimeric polypeptide comprisesthe extracellular hormone receptor ligand binding domain, anextracellular antigen-binding domain, and the transmembrane domain. 32.The single-chain chimeric polypeptide of claim 12, wherein, when goingin the C-terminal to N-terminal direction, the single-chain chimericpolypeptide comprises the extracellular hormone receptor ligand bindingdomain, an extracellular antigen-binding domain, and the transmembranedomain.